WO2013051704A1 - Glucose déshydrogénase - Google Patents

Glucose déshydrogénase Download PDF

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WO2013051704A1
WO2013051704A1 PCT/JP2012/075999 JP2012075999W WO2013051704A1 WO 2013051704 A1 WO2013051704 A1 WO 2013051704A1 JP 2012075999 W JP2012075999 W JP 2012075999W WO 2013051704 A1 WO2013051704 A1 WO 2013051704A1
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seq
amino acid
glucose dehydrogenase
acid sequence
glucose
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PCT/JP2012/075999
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Japanese (ja)
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本田　通済
さやか 滝
涼 竹中
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池田食研株式会社
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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
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • 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

Definitions

  • the present invention relates to a glucose dehydrogenase and a production method thereof, a glucose measurement method using glucose dehydrogenase, a glucose measurement reagent composition, and a glucose measurement biosensor.
  • ⁇ Rapid and accurate measurement of blood glucose concentration is important in diagnosing diabetes.
  • the enzymatic method is superior in terms of specificity and safety.
  • electrochemical biosensors are advantageous from the viewpoints of reducing the amount of specimen, reducing the measurement time, and reducing the size of the apparatus.
  • Glucose oxidase is known as an enzyme that can be used in such a biosensor.
  • glucose oxidase has a problem that measurement errors occur due to dissolved oxygen in blood
  • several glucose dehydrogenases have been developed.
  • flavin-binding glucose dehydrogenase does not require the addition of coenzymes, is not affected by dissolved oxygen, and has excellent substrate specificity. It has attracted attention (Patent Documents 1 to 4).
  • An object of the present invention is to provide a glucose dehydrogenase useful as an enzyme for a glucose biosensor, a glucose biosensor using the same, a method for identifying and isolating the glucose dehydrogenase gene, and producing a recombinant enzyme efficiently. Is to provide.
  • the present inventor paid attention to a glucose dehydrogenase derived from a filamentous fungus, isolated a glucose dehydrogenase gene derived from Botryotinia fukeliana, and further introduced the recombinant vector into microorganisms such as Escherichia coli, filamentous fungus, and yeast.
  • a recombinant glucose dehydrogenase was produced, and its enzymatic activity, amino acid sequence, availability to glucose sensor, etc. were studied.
  • the enzyme having the amino acid sequence represented by SEQ ID NO: 51 derived from Botryotinia fukeliana was supposed to be glucose oxidase (GOD) (Non-patent Document 1), but the recombinant enzyme obtained by the present invention was The present inventors have found that it is not a glucose oxidase but a flavin-binding glucose dehydrogenase, and its amino acid sequence is shown by SEQ ID NO: 5, unlike the conventionally estimated amino acid sequence. Therefore, it has been found that the flavin-binding glucose dehydrogenase obtained by the present invention is useful as an enzyme for a glucose biosensor because it is not affected by dissolved oxygen. Furthermore, among various recombinant enzymes, glycopolypeptides obtained from transformed eukaryotic cells are more useful as biosensor enzymes than polypeptides obtained from transformed E. coli without sugar chains. As a result, the present invention was completed.
  • a biosensor for glucose measurement characterized by using a flavin-binding glucose dehydrogenase comprising the following glycopeptide of (e) or (f): (E) a glycopolypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5, (F) a glycopolypeptide comprising an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 5 and having glucose dehydrogenase activity.
  • Glucose measuring reagent composition containing a flavin-binding glucose dehydrogenase comprising the following glycopolypeptide (e) or (f): (E) a glycopolypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5, (F) a glycopolypeptide comprising an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 5 and having glucose dehydrogenase activity.
  • a method for measuring glucose comprising using a flavin-binding glucose dehydrogenase comprising the following glycopolypeptide (e) or (f): (E) a glycopolypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5, (F) a glycopolypeptide comprising an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 5 and having glucose dehydrogenase activity.
  • Flavin-binding glucose dehydrogenase comprising the following glycopolypeptide (e) or (f): (E) a glycopolypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5, (F) a glycopolypeptide comprising an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 5 and having glucose dehydrogenase activity.
  • F a glycopolypeptide comprising an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 5 and having glucose dehydrogenase activity.
  • the flavin-binding glucose dehydrogenase according to [4] which is expressed in a transformed eukaryotic cell using a recombinant glucose dehydrogenase gene derived from Botryotinia fukeliana.
  • polynucleotide (a) or (b) The following polynucleotide (a) or (b): (A) a polynucleotide comprising the base sequence represented by SEQ ID NO: 4 or SEQ ID NO: 6, (B) a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the polynucleotide comprising the base sequence (a) under a stringent condition and encodes a polypeptide having glucose dehydrogenase activity.
  • A a polynucleotide comprising the base sequence represented by SEQ ID NO: 4 or SEQ ID NO: 6
  • B a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to the polynucleotide comprising the base sequence (a) under a stringent condition and encodes a polypeptide having glucose dehydrogenase activity.
  • a recombinant vector comprising the polynucleotide according to any one of [6] to [9].
  • the glycopolypeptide of the present invention is a flavin-binding glucose dehydrogenase and is useful for a glucose biosensor that can accurately measure glucose without being affected by dissolved oxygen.
  • the polynucleotide of the present invention it is possible to produce a glucose dehydrogenase having excellent properties such as excellent substrate recognizability to glucose and low activity against maltose in a homogeneous and large-scale production. .
  • glucose dehydrogenase refers to catalyzing the reaction of dehydrogenating (oxidizing) the hydroxyl group at the 1-position of glucose in the presence of an electron acceptor, and acting on maltose with respect to its action on glucose It means a soluble protein having a property of 5% or less, and the enzyme is characterized by the following properties.
  • Flavin adenine dinucleotide (FAD) as a coenzyme 2) substantially no oxygen as an electron acceptor, 3) At a substrate concentration of 333 mM, when the activity on glucose is 100%, the activity on maltose is 5% or less, and 4) the molecular weight of the polypeptide of the enzyme protein is 60-70 kDa.
  • the molecular weight of the polypeptide of the enzyme protein is the molecular weight when the glycosylated recombinant enzyme expressed in eukaryotic cells is subjected to sugar chain cleavage treatment and subjected to SDS-polyacrylamide electrophoresis.
  • Recombinant enzymes expressed in eukaryotic cells can have a molecular weight of about 80 to 200 kDa because the amount of glycosylation varies depending on the host, culture conditions, and the like. It is preferably 90 to 200 kDa, more preferably 90 to 150 kDa, still more preferably 90 to 120 kDa.
  • “glycopolypeptide” refers to a polypeptide to which a sugar chain is added.
  • the polynucleotide of the present invention is the following polynucleotide (a) or (b).
  • polynucleotide of the present invention is a polynucleotide encoding the following polypeptide (c) or (d).
  • C a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 5
  • D A polypeptide comprising an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence (c) and having glucose dehydrogenase activity.
  • polynucleotide (b) or polypeptide (d) examples include those having a base sequence or amino acid sequence having at least 80% homology with the base sequence (a) or amino acid sequence (c).
  • the base sequence or amino acid sequence having at least 80% homology shows at least 80% identity over the entire length of the reference sequence to be compared, preferably at least 85%, more preferably at least 90%. %, More preferably each sequence having at least 95% identity.
  • sequence identity percentage can be calculated using publicly available or commercially available software with an algorithm that compares the reference sequence as a query sequence. For example, GeneDoc or GENETYX (Software Development Co.) 'S Maximum Matching can be used, and these can be used with default parameters.
  • hybridization under stringent conditions for hybridization between polynucleotides include, for example, 50% formamide, 5 ⁇ SSC (150 mM sodium chloride, 15 mM trisodium citrate, 10 mM sodium phosphate, 1 mM ethylenediaminetetraacetic acid, pH 7.2), 5 ⁇ Denhardt's solution, 0.1% SDS, 10% dextran sulfate and 100 ⁇ g / mL denatured salmon sperm DNA, incubated at 42 ° C., filter Can be exemplified by washing at 42 ° C. in 0.2 ⁇ SSC.
  • polynucleotide means a nucleoside phosphate ester (ATP (adenosine triphosphate), GTP (guanosine triphosphate), CTP (purine or pyrimidine linked to a sugar).
  • ATP adenosine triphosphate
  • GTP guanosine triphosphate
  • CTP purine or pyrimidine linked to a sugar
  • Cytidine triphosphate UTP (uridine triphosphate); or dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidine triphosphate), dTTP (deoxythymidine triphosphate)
  • UTP uridine triphosphate
  • dATP deoxyadenosine triphosphate
  • dGTP deoxyguanosine triphosphate
  • dCTP deoxycytidine triphosphate
  • dTTP deoxythymidine triphosphate
  • Polypeptide means a molecule composed of 30 or more amino acid residues joined together by amide bonds (peptide bonds) or unnatural residue linkages.
  • “Glycopolypeptide” means a molecule in which a sugar chain is added to the polypeptide.
  • the most specific embodiment of the polynucleotide (gene) of the present invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6.
  • a polynucleotide which is a chromosomal DNA represented by SEQ ID NO: 6 is prepared by preparing a chromosomal DNA library from, for example, Botryotinia fukeliana strain, and the amino acid sequence of Aspergillus terreus-derived glucose dehydrogenase described in Patent Document 2 And a plurality of oligonucleotide probes prepared based on the amino acid sequence of glucose dehydrogenase derived from Aspergillus oryzae or other amino acid sequences of glucose dehydrogenase by the method known to those skilled in the art. It can be obtained by screening the rally.
  • the labeling of the probe can be performed by any method known to those skilled in the art, for example, the radioisotope (RI) method or the non-RI method, but the non-RI method is preferably used.
  • the non-RI method include a fluorescence labeling method, a biotin labeling method, a chemiluminescence method, and the like, but it is preferable to use a fluorescence labeling method.
  • the fluorescent substance those capable of binding to the base moiety of the oligonucleotide can be appropriately selected and used.
  • cyanine dyes eg, Cy Dye TM series Cy3, Cy5, etc.
  • rhodamine 6G reagent N-acetoxy-N2 -Acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) and the like can be used.
  • a polynucleotide that is a cDNA represented by SEQ ID NO: 4 is obtained, for example, after obtaining a target polynucleotide containing an intron from chromosomal DNA, as specifically described in the Examples of the present specification,
  • it can be obtained by various PCR methods known to those skilled in the art using a set of oligonucleotide primers (probes) prepared above using a cDNA library as a template.
  • it can also be obtained by the RT-PCR method using total RNA or mRNA extracted from Botryotinia fukeliana strain as a template.
  • the final concentration of primers used in PCR is from about 0.1 to about 1 ⁇ M.
  • commercially available software such as Oligo TM (National Bioscience Inc. (USA)), GENETYX (Software Development Co., Ltd.) and the like can also be used.
  • Such an oligonucleotide probe or oligonucleotide primer set can also be prepared, for example, by cleaving cDNA, which is a polynucleotide of the present invention, with an appropriate restriction enzyme.
  • the polynucleotide of the present invention is a polynucleotide having a base sequence encoding a signal sequence upstream of the polynucleotide described in [6] or [7].
  • the base sequence encoding the signal sequence may be any base sequence encoding a signal sequence capable of efficiently secreting a glycopolypeptide having glucose dehydrogenase activity, for example, a base sequence encoding a signal sequence of a eukaryotic cell-derived protein.
  • a base sequence encoding a signal sequence of a protein derived from the same gene as the host at the time of production of the recombinant protein is more preferable, a base sequence encoding a signal sequence of a protein derived from a filamentous fungus is more preferable, More preferred is a base sequence encoding a signal sequence. Further examples include a base sequence encoding a signal sequence of a protein derived from the same gene as the host transformed with the polynucleotide, and a base sequence encoding a signal sequence of glucose dehydrogenase.
  • the signal sequence encoded by the base sequence can be obtained by, for example, comparing the Aspergillus terreus-derived glucose dehydrogenase sequence described in Patent Document 2 with the amino acid sequence of the secreted protein, or a signal sequence prediction site (Signal p: http: / /www.cbs.dtu.dk/services/SignalP/), which may be deduced from the amino acid sequence of the secreted protein, and further may be a signal sequence in which one to several amino acids are substituted, deleted or added.
  • the sequences described in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14 can be exemplified. These sequences or sequences obtained by deleting several sequences of these sequences may be combined as appropriate.
  • polynucleotide having the base sequence encoding the signal sequence examples include SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and sequence.
  • part which codes the protein which has glucose dehydrogenase activity may contain the intron.
  • the polynucleotides (a) to (d) can be prepared, for example, by modifying the above-mentioned glucose dehydrogenase cDNA derived from Botryotinia fukeliana by a known mutation introduction method or mutation introduction PCR method. it can. Furthermore, it can be obtained by a probe hybridization method using an oligonucleotide prepared based on the nucleotide sequence information of SEQ ID NO: 1 from a chromosomal DNA of a Botryotinia fukeliana strain other than the NBRC7185 strain or a cDNA library thereof. In the hybridization, the above-mentioned polynucleotide can be obtained by variously changing the stringent conditions.
  • Stringent conditions are defined by the salt concentration in the hybridization and washing steps, the concentration of organic solvent (formaldehyde, etc.), temperature conditions, and the like.
  • concentration of organic solvent formaldehyde, etc.
  • temperature conditions and the like.
  • the recombinant vector of the present invention is a cloning vector or an expression vector, and an appropriate one is used depending on the type of polynucleotide as an insert, the purpose of use, and the like.
  • an appropriate one is used depending on the type of polynucleotide as an insert, the purpose of use, and the like.
  • it is suitable for expression vectors for in vitro transcription and eukaryotic cells such as yeast, filamentous fungi, insect cells and mammalian cells. Expression vectors can also be used.
  • transformed cells of the present invention eukaryotic cells such as yeast, filamentous fungi, insect cells and mammalian cells can be used. These transformed cells can be prepared by introducing a recombinant vector into cells by a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method. Specific examples of the recombinant vector and the transformed cell include the recombinant vector shown in the examples below, and the transformed filamentous fungi and transformed yeast using this vector.
  • glucose dehydrogenase When glucose dehydrogenase is expressed in eukaryotic cells for production, the polynucleotide is inserted into an eukaryotic expression vector having a promoter, a splicing region, a poly (A) addition site, etc.
  • glucose dehydrogenase can be produced in eukaryotic cells. It can be maintained in the cell in a state like a plasmid, or it can be maintained in a chromosome.
  • expression vectors include pKA1, pCDM8, pSVK3, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pYE82 and the like.
  • glucose dehydrogenase poly- hydrase can be used as a fusion protein to which various tags such as His tag, FLAG tag, GFP are added.
  • Peptides can also be expressed.
  • mammalian cultured cells such as monkey kidney cells COS-7 and Chinese hamster ovary cells CHO, budding yeast, fission yeast, filamentous fungi, silkworm cells, Xenopus egg cells, etc. are generally used, but glucose dehydrogenation is used.
  • Any eukaryotic cell may be used as long as it can express the enzyme.
  • a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method can be used.
  • the inserted gene for production when expressed in eukaryotic cells may or may not contain introns.
  • SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, sequence A gene sequence containing the signal sequence described in SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, or SEQ ID NO: 48 is preferred, but when there is a secretory signal sequence on the vector side, the gene sequence described in SEQ ID NO: 4 or SEQ ID NO: 6
  • a polynucleotide in which a start codon ATG is added to the gene sequence described in SEQ ID NO: 4 or 6 may be inserted. Secretory production is preferred.
  • the expression efficiency of the recombinant glucose dehydrogenase of the present invention is high, preferably at least 20 U per mL of culture medium, more preferably at least 50 U per mL of culture medium, more preferably at least 80 U per mL of culture medium, particularly preferably Is at least 100 U per mL of culture and most preferably at least 120 U per mL of culture.
  • the recombinant enzyme can be stably and highly expressed.
  • the specific activity of the crude enzyme obtained from the culture is high and there are few contaminating proteins, the purification process is simple, the purification cost is reduced, and the purified enzyme can be obtained in high yield.
  • a known separation operation can be performed in combination. For example, treatment with denaturing agents and surfactants such as urea, heat treatment, pH treatment, ultrasonic treatment, enzyme digestion, salting out and solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, etc.
  • denaturing agents and surfactants such as urea, heat treatment, pH treatment, ultrasonic treatment, enzyme digestion, salting out and solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, etc.
  • Electrophoresis ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, affinity chromatography (including methods using tag sequences, polyclonal antibodies specific to glucose dehydrogenase, and methods using monoclonal antibodies) , Etc.
  • glucose dehydrogenase can be obtained by a recombinant DNA technique using the polynucleotide (cDNA or its translation region) of the present invention.
  • a recombinant DNA technique using the polynucleotide (cDNA or its translation region) of the present invention.
  • it is possible to prepare glucose dehydrogenase in vitro by preparing RNA from a vector having the polynucleotide by in vitro transcription and performing in vitro translation using this as a template.
  • a polynucleotide is recombined into an appropriate expression vector by a known method, a large amount of glucose dehydrogenase encoded by the polynucleotide can be expressed in eukaryotic cells such as yeast, mold, insect cells, and mammalian cells. I can do things.
  • a polynucleotide having the same amino acid sequence corresponding to the host but having optimized codon usage may be introduced.
  • a recombinant vector is prepared by inserting the polynucleotide into a vector having a promoter to which RNA polymerase can bind, and this vector is converted into RNA corresponding to the promoter.
  • Glucose dehydrogenase can be produced in vitro by adding it to an in vitro translation system such as a rabbit reticulocyte lysate containing a polymerase or a wheat germ extract.
  • promoters to which RNA polymerase can bind include T3, T7, SP6 and the like.
  • vectors containing these promoters include pKA1, pCDM8, pT3 / T718, pT7 / 319, and pBluescript II.
  • the glucose dehydrogenase of the present invention is a glucose dehydrogenase comprising the following sugar polypeptide (e) or (f).
  • the most specific embodiment of the glucose dehydrogenase of the present invention is a glycopolypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5.
  • it may be a glycopolypeptide consisting of an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 5.
  • the number is preferably 100 or less, more preferably 80 or less, still more preferably 60 or less, still more preferably 40 or less, still more preferably 20 or less, still more preferably 10 or less, and still more preferably 5 or less.
  • glucose dehydrogenase having a glycopolypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 or SEQ ID NO: 49 Can be illustrated.
  • the glucose dehydrogenase of the present invention has at least 80% homology with the amino acid sequence of SEQ ID NO: 5, more preferably 85% or more homology, more preferably 90% or more homology, more preferably 95 %, More preferably 97% or more of an amino acid sequence having a homology of 97% or more, and a glycopolypeptide having glucose dehydrogenase activity.
  • the feature of the glucose dehydrogenase of the present invention is (e) a glycopeptide consisting of the amino acid sequence represented by SEQ ID NO: 5 or its analog (f).
  • SEQ ID NO: 5 is a sequence in which 19 amino acids on the N-terminal side are deleted from the sequence of SEQ ID NO: 2, unlike the conventionally estimated sequence.
  • SEQ ID NO: 2 the N-terminal 19 amino acid sequence was found to be a signal sequence. Therefore, the glucose dehydrogenase of the present invention is a novel enzyme having a novel amino acid sequence.
  • the glucose dehydrogenase of the present invention is a glycopeptide.
  • the positions where N-type sugar chains are added are the 27th, 44th, 47th, 73th, 173rd, 222th, 256th and 290th asparagines of SEQ ID NO: 5 (this is the same as SEQ ID NO: 2). 46th, 63rd, 66th, 92nd, 192nd, 241st, 275th, and 309th asparagine).
  • the glucose dehydrogenase of the present invention is superior as an enzyme for measuring glucose compared to an enzyme having the same amino acid sequence and no sugar chain. That is, when a biosensor was constructed using an enzyme having the same amino acid sequence as that of the glucose dehydrogenase of the present invention and having no sugar chain, and the glucose concentration was measured, the glucose dehydrogenase of the present invention was more sensitive. Was twice as high.
  • glucose dehydrogenase of the present invention was superior in thermal stability as compared with an enzyme having no sugar chain.
  • the glucose dehydrogenase of the present invention is particularly useful as an enzyme for measuring glucose because it has a novel amino acid sequence and a sugar chain.
  • the glucose dehydrogenase of the present invention is an enzyme that catalyzes a reaction for dehydrogenating glucose in the presence of an electron acceptor
  • the glucose dehydrogenase is not particularly limited as long as the change due to this reaction can be used.
  • it can be used in the medical field and clinical field such as measuring and measuring reagents in glucose-containing samples, and used as a reagent for erasing, and is capable of producing substances using flavin-binding glucose dehydrogenase. Can also be used.
  • the glucose dehydrogenase of the present invention can be used in a glucose measurement reagent composition.
  • the measurement reagent composition of the present invention comprises a glucose dehydrogenase comprising the glycopolypeptide of the present invention, bovine serum albumin (BSA) or ovalbumin, a saccharide or sugar alcohol having no activity with the enzyme, a carboxyl group-containing compound
  • BSA bovine serum albumin
  • Other appropriate components known to those skilled in the art such as a heat stabilizer selected from the group consisting of alkaline earth metal compounds, ammonium salts, sulfates or proteins, or buffers, etc.
  • the thermal stability and storage stability of can be improved.
  • the measurement reagent composition can contain a known substance that suppresses the influence of a contaminant substance that affects the measurement present in the test sample.
  • the composition may contain a buffer so that the enzyme has a pH at which the enzyme is likely to act when dissolved.
  • the glucose dehydrogenase of the present invention can be used in a biosensor as a glucose sensor for measuring the glucose concentration in a sample solution.
  • the biosensor of the present invention may be any sensor that uses glucose dehydrogenase comprising the glycopolypeptide of the present invention as an enzyme in the reaction layer.
  • the biosensor includes a step of forming an electrode system including at least a working electrode and a counter electrode using a method such as screen printing or vapor deposition on an insulating substrate, a liquid enzyme and an electron acceptor at the same time or each plate.
  • Manufactured by a manufacturing process including a process of dropping on top. In the production process, it is necessary that the temperature of the liquid enzyme before dropping is preferably not left at 45 ° C.
  • Dropping means placing or placing a liquid enzyme on a plate.
  • the measurement reagent is dissolved and the enzyme reacts with the substrate, and the electron acceptor is reduced accordingly.
  • the reduced electron acceptor is oxidized electrochemically.
  • the biosensor can measure the substrate concentration in the sample solution from the obtained oxidation current value. If the glucose dehydrogenase of the present invention is used, it is possible to measure up to at least 40 mM glucose.
  • a biosensor capable of measuring more than 5 mM and at least 40 mM can be obtained. That is, it is possible to obtain a biosensor that can measure to an arbitrary concentration from 5 mM to at least 40 mM. In addition, it is possible to construct a biosensor that detects color intensity or pH change.
  • Electrode acceptor of the biosensor As the electron acceptor of the biosensor, a substance excellent in electron transfer capability can be used.
  • Substances that excel in electron transfer are chemical substances generally called “electron mediators,” “mediators,” or “redox mediators,” and protein-type electron mediators.
  • the electron carriers and redox mediators listed in JP-T-2002-526759 may be used.
  • the glucose dehydrogenase of the present invention can be used in a biobattery.
  • the biobattery according to the present invention includes an anode electrode that performs an oxidation reaction and a cathode electrode that performs a reduction reaction, and includes an electrolyte layer that separates the anode and the cathode as necessary.
  • An enzyme electrode containing the above-described electron mediator and the enzyme of the present invention is used as an anode electrode, and electrons generated by oxidizing the substrate are taken out to the electrode and protons are generated.
  • an enzyme generally used for the cathode electrode may be used on the cathode side.
  • laccase laccase, ascorbate oxidase or bilirubin oxidase is used, and the proton generated on the anode side is reacted with oxygen. Generate water.
  • an electrode an electrode generally used for a bio battery such as carbon, gold, or platinum can be used.
  • the amount of decrease per minute ( ⁇ A600) in absorbance at 600 nm accompanying the progress of the enzyme reaction was measured for 5 minutes from the start of the reaction, and the GLD activity was calculated from the linear portion according to Equation 1. At this time, the GLD activity was defined as 1 U for the amount of enzyme that reduces 1 ⁇ mol of DCIP per minute at 37 ° C. and pH 6.0.
  • 3.0 is the volume of the reaction reagent + enzyme solution (mL)
  • 10.8 is the molar extinction coefficient of DCIP (mM ⁇ 1 cm ⁇ 1 ) at pH 6.0
  • 1.0 is the optical path of the cell Length (cm)
  • 0.05 is the volume of the enzyme solution (mL)
  • ⁇ A600 blank is the decrease in absorbance per minute at 600 nm when the reaction is started by adding the solution used for enzyme dilution instead of the enzyme solution.
  • the amount, df represents the dilution factor.
  • the enzyme is preferably diluted as appropriate so that the final concentration is preferably 0.2 to 0.9 mg / mL.
  • the protein concentration in the present invention was determined by using bovine serum albumin (BSA, manufactured by Wako Pure Chemical Industries, Ltd.) using Bio-Rad Protein Assay, which is a protein concentration measurement kit that can be purchased from Nippon Bio-Rad Co., Ltd. , For biochemistry) can be calculated from a calibration curve created as a standard substance.
  • BSA bovine serum albumin
  • Bio-Rad Protein Assay which is a protein concentration measurement kit that can be purchased from Nippon Bio-Rad Co., Ltd. , For biochemistry
  • Example 1 (Confirmation of glucose dehydrogenase (BfGLD) activity derived from Botryotinia fukeliana) Potato dextrose broth (manufactured by Difco) 100 mL of 2.4% medium was placed in a 500 mL Sakaguchi flask, capped, and autoclaved at 121 ° C. for 20 minutes.
  • BfGLD glucose dehydrogenase
  • the cooled liquid medium was inoculated with Botryotinia fukeliana strain NBRC7185, and the cells were cultured at 25 ° C. for 216 hours under aeration and agitation. As a result, 0.21 U / mL of the culture was added to the culture supernatant. / ML glucose dehydrogenase activity was confirmed. Glucose oxidase (GOD) activity was not detected. GOD activity was measured by the following method.
  • the GOD activity was defined as 1 U for the amount of enzyme that produces 1 ⁇ mol of hydrogen peroxide per minute at 37 ° C. and pH 7.0.
  • 3.0 is the amount of the reaction reagent + enzyme solution (mL)
  • 10.66 is the molar extinction coefficient (mM ⁇ 1 cm ⁇ 1 ) under the present measurement conditions
  • 0.5 is 1 mol of peroxide.
  • the amount of quinone dye produced relative to the amount of hydrogen produced 1.0 is the optical path length of the cell (cm)
  • 0.05 is the amount of the enzyme solution (mL)
  • ⁇ A500 blank is the amount of the enzyme solution diluted with the enzyme solution.
  • the amount of increase in absorbance at 500 nm per minute when the reaction is started by addition, df represents the dilution factor.
  • Example 2 (Expression of glucose dehydrogenase (BfGLD) derived from recombinant Botryotinia fukeliana by eukaryotic cells)
  • Cell culture Glucose (manufactured by Nacalai) 1% (W / V), defatted soybean (manufactured by Showa Sangyo Co., Ltd.) 2% (W / V), corn steep liquor (manufactured by Sanei Sugar Chemical Co., Ltd.) 0.5% ( W / V), magnesium sulfate heptahydrate (manufactured by Nacalai Co., Ltd.) 0.1% (W / V) and water were adjusted to pH 6.0, and 100 mL was placed in a 500 mL Sakaguchi flask.
  • BfGLD gene was PCR amplified using the DNA acquired in (2) as a template.
  • Primers analyze a common sequence from a plurality of GLD gene sequences that have already been elucidated by the present inventors, design a degenerate primer based on the common sequence, obtain a gene fragment, and finally the following primers : Using the primer1F (SEQ ID NO: 15) and primer2R (SEQ ID NO: 16), a DNA fragment of about 1.8 kbp containing the entire length of the target GLD gene (including intron) was obtained.
  • primer1F (TGACCAATTCCGCAGCTCGTCAAA) ATGTATCGTTTACTCTCTACATTTG (SEQ ID NO: 15) (In parentheses: transcription enhancer) primer2R: ((GCTATCCTGTTACGCTTCTAGA)) GCATGC CTAAATGTCCTCCTTGATCAAATCT (SEQ ID NO: 16) (In parentheses: pSENS vector sequence, underlined: restriction enzyme site (SphI)) primer3F: ((CCGTCCTCCAAGTTA)) GTCGAC (TGACCAATTCCGCAGCTCGTCAAA) (SEQ ID NO: 17) (In parentheses: pSENS vector sequence, underlined: restriction enzyme site (SalI), parentheses: transcription enhancing factor)
  • Illustra plasma-prep MINI Flow Kit manufactured by GE Healthcare
  • Illustra plasma-prep MINI Flow Kit manufactured by GE Healthcare
  • a BfGLD gene SEQ ID NO: 3 containing an intron could be confirmed.
  • Recombinant mold (Aspergillus oryzae) producing BfGLD was prepared according to the method described in Technology, Katsuya Gomi, Brewery, 494-502, 2000), and the obtained recombinant strain was purified with Czapek-Dox solid medium .
  • Aspergillus oryzae NS4 strain was used as a host to be used. This strain is bred at a brewery laboratory in 1997 as disclosed in known document 2, and is used for analysis of transcription factors, breeding of high-producing strains of various enzymes, etc. Is available.
  • the transformant obtained in (5) was inoculated into this cooled liquid medium, and cultured with shaking at 30 ° C. for 3 to 4 days. After completion of the culture, the supernatant was collected by centrifugation and used as a sample with recombinant BfGLD sugar chains (Sc (+) BfGLD).
  • glucose dehydrogenase activity (U / mL) of recombinant Sc (+) BfGLD was measured according to the enzyme activity measurement method described above, it was 133 U / mL on the third day and 98 U / mL on the fourth day per 1 mL of the culture solution. The glucose dehydrogenase activity was confirmed. Glucose oxidase activity could not be confirmed.
  • the recombinant BfGLD of the present invention had a reactivity with maltose of 1.3% when the substrate concentration was 333 mM and the activity against D-glucose was 100%.
  • the culture solution is filtered through a filter cloth, and the collected filtrate is centrifuged to collect the supernatant, and further filtered through a membrane filter (10 ⁇ m, manufactured by Advantech) to collect the culture supernatant, and the molecular weight cutoff
  • a membrane filter (10 ⁇ m, manufactured by Advantech) to collect the culture supernatant, and the molecular weight cutoff
  • the resultant was concentrated with an 8,000 ultrafiltration membrane (Millipore) to obtain a crude enzyme solution.
  • the specific activity of the crude enzyme solution was 1,170 U / mg.
  • the crude enzyme solution was adjusted to a 50% saturated ammonium sulfate solution (pH 5.0), allowed to stand overnight at 4 ° C., and then centrifuged to collect the supernatant.
  • the supernatant was passed through a TOYOPEARL Butyl-650C (manufactured by Tosoh Corp.) column ( ⁇ 7.5 cm ⁇ 17.7 cm) pre-equilibrated with 50 mM sodium acetate buffer (pH 5.0) containing 50% saturated ammonium sulfate. Enzyme was adsorbed.
  • the column was washed with the same buffer, and then the enzyme was eluted by a gradient elution method from the buffer to 50 mM sodium acetate buffer (pH 5.0) to collect the active fraction.
  • the collected active fraction is concentrated with an ultrafiltration membrane, desalted, equilibrated with 1 mM sodium acetate buffer (pH 5.0), and DEAE Cellufine A-500m (Chisso) pre-equilibrated with the same buffer.
  • the enzyme was adsorbed by passing through a column ( ⁇ 6.0 cm ⁇ 21.9 cm). After washing the column with the same buffer, the enzyme was eluted by a gradient elution method from the buffer to a 200 mM sodium acetate buffer (pH 5.0) to collect the active fraction.
  • the collected active fraction was concentrated with an ultrafiltration membrane having a fractional molecular weight of 8,000, and then replaced with water to obtain a purified enzyme of a sample with a recombinant BfGLD sugar chain (Sc (+) BfGLD).
  • the specific activity of the purified enzyme was 2,100 U / mg.
  • the recombinant BfGLD before and after sugar chain cleavage was subjected to SDS-polyacrylamide electrophoresis for confirmation. Specifically, 5 ⁇ L of recombinant Sc (+) BfGLD and 5 ⁇ L of 0.4 M potassium phosphate buffer (pH 6.0) containing 1% SDS and 2% ⁇ -mercaptoethanol were mixed and heat-treated at 100 ° C.
  • a gene encoding the C-terminus from the predicted N-terminus of wild-type BfGLD can be amplified as follows: primer4F_BfGLD_Sig ( ⁇ ) / Sac PCR was performed using a primer set of (SEQ ID NO: 18) and primer5R_BfGLD / Hind (SEQ ID NO: 19).
  • primer4F_BfGLD_Sig ( ⁇ ) / Sac PCR was performed using a primer set of (SEQ ID NO: 18) and primer5R_BfGLD / Hind (SEQ ID NO: 19).
  • Obtaining a polynucleotide that does not contain the gene sequence that encodes the expected signal sequence can be obtained when the recombinant protein is expressed in E. coli using a foreign gene that includes the gene sequence that encodes the secretory signal sequence.
  • the predicted N-terminus of wild-type BfGLD is the sequence identity between the full-length amino acid sequence of BfGLD (SEQ ID NO: 2) and Aspergillus terreus-derived glucose dehydrogenase (AtGLD) described in SEQ ID NO: 2 of WO 2006/101239 pamphlet. From the 20th and subsequent amino acid sequences of SEQ ID NO: 2 of WO 2006/101239, which is the N terminus of wild-type AtGLD, the N-terminal of wild-type BfGLD is the 17th and subsequent amino acid sequences described in SEQ ID NO: 1. Predicted.
  • primer4F_BfGLD_Sig (-) / Sac: AAA GAGCTC GAGCACCGACTCTACCTTAAA (SEQ ID NO: 18) (Underlined: restriction enzyme site (SacI)) primer5R_BfGLD / Hind: CCC AAGCTT CTAAATGTCCTCCTTGATC (SEQ ID NO: 19) (Underlined part: restriction enzyme site (HindIII))
  • primer set of primer4F_BfGLD_Sig (-) / Sac (SEQ ID NO: 18) and the following primer6R_Bf_up (int) (SEQ ID NO: 20) or the following primer7F_Bf_ ( Int) down (SEQ ID NO: 21) and primer5R_BfGLD / Hind (SEQ ID NO: 19) were used for PCR amplification under the PCR conditions.
  • primer6R_Bf_up (int): GGGTGTATGCCATTCCATTGATAGTACTAGTCCCT (SEQ ID NO: 20) primer7F_Bf_ (int) down: GAATGGCATACACCCGAGCCGAAGAT (SEQ ID NO: 21)
  • primer7F_Bf_ (int) down: GAATGGCATACACCCGAGCCGAAGAT (SEQ ID NO: 21)
  • the obtained two amplified sequences were put into one PCR amplification tube, and PCR-amplified using the primer set of primer4F_BfGLD_Sig (-) / Sac (SEQ ID NO: 18) and primer5R_BfGLD / Hind (SEQ ID NO: 19) under the PCR conditions, A DNA fragment in which a primer addition sequence was added to the 16AA ( ⁇ ) BfGLD gene not containing an intron and not containing a 48 bp base sequence encoding the expected signal sequence was obtained.
  • the preculture solution 50 mL of the preculture solution is inoculated into a 2 L jar fermenter containing 1.5 L of LB liquid medium containing 50 ⁇ g / mL ampicillin, cultured at 37 ° C., and the OD600 of the culture solution is about 0.4 to 0.5.
  • 1 mM of IPTG was added and cultured for 25.5 hours under aeration and stirring conditions.
  • the cells collected by centrifugation were suspended in 20 mM potassium phosphate buffer (pH 6.0), and the cells were disrupted using an ultrasonic crusher and then centrifuged to prepare a cell-free extract.
  • the specific activity of the cell-free extract was 0.0182 U / mg.
  • the cell-free extract was dialyzed in 20 mM potassium phosphate buffer (pH 7.5), passed through a DEAE-Cellulofine A-500 column equilibrated with the same buffer, and the eluate was collected.
  • the eluate was dialyzed in 5 mM potassium phosphate buffer (pH 7.5) and passed through a DEAE-Cellulofine A-500 column equilibrated with the same buffer to adsorb the enzyme. After washing the column with the same buffer, the active fraction was collected by eluting the enzyme by the gradient elution method from the buffer to the same buffer containing 0.3 M potassium chloride.
  • the active fraction was concentrated with an ultrafiltration membrane having a fractional molecular weight of 10,000, desalted and then replaced with 20 mM potassium phosphate buffer (pH 6.0) to obtain a purified enzyme of Sc ( ⁇ ) BfGLD. Furthermore, when recombinant Sc (-) BfGLD was subjected to SDS-polyacrylamide electrophoresis and the molecular weight was determined from the molecular weight marker, recombinant Sc (-) BfGLD of about 60 kDa could be confirmed.
  • Example 3 Comparison with known sequence of recombinant BfGLD
  • the amino acid sequence of BfGLD including the signal sequence portion was as shown in SEQ ID NO: 2, but when BLAST search was performed using this sequence, it surprisingly matched the amino acid sequence of glucose oxidase derived from Botriotinia fukeliana .
  • BfGLD obtained in the present invention had glucose dehydrogenase activity as described in Example 2, (6), and no glucose oxidase activity was observed.
  • the recombinant BfGLD of the present invention has a molecular weight in SDS-polyacrylamide electrophoresis of 90-100 kDa for the enzyme before sugar chain cleavage and 60-70 kDa for the enzyme after sugar chain cleavage when recombined in eukaryotic cells.
  • Glucose dehydrogenase For example, publicly known document 6 (Protein Nucleic Acid Enzyme (2004) 49 (5), 625-633, Review) describes that xanthine dehydrogenase is converted to xanthine oxidase by partial degradation with a protease. .
  • Botryotinia fukeliana-derived glucose dehydrogenase and the Botryotinia fukeliana-derived glucose dehydrogenase of the present invention are the same as the amino acid sequence deduced from the gene sequence.
  • Glucose oxidase has a subunit molecular weight of 35 kDa and has glucose oxidase activity, and is completely different in structure and function. The difference between the two is that after taking a three-dimensional structure, some modification such as degradation by protease is performed. I guess it depends on whether or not . In particular, in the enzyme recombined with Aspergillus, activity conversion due to protease modification was not observed. Therefore, recombinant production with Aspergillus seems to be a meaningful method for efficiently producing recombinant BfGLD.
  • the GC electrode was connected to potentiostat BAS100B / W (manufactured by BAS), the solution was stirred at 37 ° C., and +500 mV was applied to the silver-silver chloride reference electrode. 5 ⁇ l of 1M D-glucose solution was added to these systems so that the final concentration was 10 mM, and the steady-state current value was measured. Furthermore, the operation of adding a 1M D-glucose solution to each final concentration and measuring the current value was repeated, and finally the current value at each glucose concentration (5, 10, 20, 30, 40 mM) was measured. . When this was plotted, a calibration curve was created (FIG. 1).
  • Example 6 (Signal sequence mutation) In the production of recombinant BfGLD by recombinant mold, the secretion signal was mutated.
  • the primers described below were synthesized, and the BfGLD gene with the signal sequence substituted was amplified by the PCR method using the plasmid vector obtained in (2) of Example 2 as a template.
  • primer8_Bf-Atsignal1-7AA-F ((CCCTGTCCCTGGCAGTGGCGGCACCTTTG)) TTTGCTGTAGCCTCTTTGGCTGCAG (SEQ ID NO: 22)
  • primer9_Bf-Atsignal2-F ((ATGTTGGGAAAGCTCTCCTTCCTCAGTGCCCTGTCCCTGGCAGTGGCGGCACCTTTG))
  • primer10_Bf-eno-Atsignal-F (TGACCAATTCCGCAGCTCGTCAAA) ((ATGTTGGGAAAGCTCCTTCCTCA))
  • primer11_Bf-infusion-F CTCCAAGTTA GTCGAC (TGACCAATTCCGCAGCTCGTCAAA)
  • primer12_Bf-infusion-R CGCTTCTAGA GCATGC CTAAATGTCCTCCTTGATCAAATC (SEQ ID NO: 26) primer13_Bf-Aosig-s
  • PCR using primer8_Bf-Atsignal1-7AA-F and primer12_Bf-infusion-R was performed using the plasmid extracted in (2) of Example 2 as a template.
  • PCR was performed using primer9_Bf-Atsignal2-F and primer12_Bf-infusion-R using the PCR product of the first step as a template.
  • PCR was performed using primer10_Bf-eno-Atsignal-F and primer12_Bf-infusion-R as the third step, using the PCR product of the second step as a template.
  • PCR was performed using primer11_Bf-infusion-F and primer12_Bf-infusion-R using the PCR product of the third step as a template as the fourth step.
  • the obtained PCR product contains an intron that encodes a sequence in which 7 amino acids (MYRLLLST) of the signal sequence portion of the full-length amino acid sequence of BfGLD (SEQ ID NO: 2) are substituted with the AtGLD signal sequence (SEQ ID NO: 10). It is a polynucleotide.
  • the sequence containing no intron of the sequence is shown as SEQ ID NO: 36 as the Atsig-7AA ( ⁇ ) BfGLD gene, and the amino acid sequence encoded by the sequence is shown in SEQ ID NO: 37.
  • PCR was performed using primer13_Bf-Aosig-short1-7AA-F and primer12_Bf-infusion-R using the plasmid extracted in (2) of Example 2 as a template.
  • PCR was performed using primer14_Bf-Aosig-short2-F and primer12_Bf-infusion-R using the PCR product of the first step as a template.
  • PCR was performed using primer17_Bf-eno-Aosignal-F and primer12_Bf-infusion-R as the third step, using the PCR product of the second step as a template.
  • PCR was performed using primer11_Bf-infusion-F and primer12_Bf-infusion-R using the PCR product of the third step as a template as the fourth step.
  • the obtained PCR product encodes a sequence in which 7 amino acids (MYRLLLST) of the signal sequence portion of the full-length amino acid sequence of BfGLD (SEQ ID NO: 2) are replaced with a predicted signal sequence (SEQ ID NO: 12) of AoGLD.
  • a polynucleotide comprising A sequence not containing an intron of the sequence is shown as SEQ ID NO: 38 as an AosigS-7AA ( ⁇ ) BfGLD gene, and an amino acid sequence encoded by the sequence is shown in SEQ ID NO: 39.
  • PCR using primer15_Bf-Aosignal1-7AA-F and primer12_Bf-infusion-R was performed using the plasmid extracted in (2) of Example 2 as a template.
  • PCR was performed using primer16_Bf-Aosignal2-F and primer12_Bf-infusion-R using the PCR product of the first step as a template.
  • PCR was performed using primer17_Bf-eno-Aosignal-F and primer12_Bf-infusion-R as the third step, using the PCR product of the second step as a template.
  • PCR was performed using primer11_Bf-infusion-F and primer12_Bf-infusion-R using the PCR product of the third step as a template as the fourth step.
  • the obtained PCR product encodes a sequence in which 7 amino acids (MYRLLLST) of the signal sequence portion of the full-length amino acid sequence of BfGLD (SEQ ID NO: 2) are replaced with the expected signal sequence (SEQ ID NO: 14) of AoGLD.
  • a polynucleotide comprising The sequence not containing an intron of this sequence is shown as SEQ ID NO: 40 as the AosigL-7AA ( ⁇ ) BfGLD gene, and the amino acid sequence encoded by the sequence is shown in SEQ ID NO: 41.
  • PCR was performed using primer18_Bf-Atsignal1-16AA-F and primer12_Bf-infusion-R using the plasmid extracted in (2) of Example 2 as a template.
  • PCR was performed using primer9_Bf-Atsignal2-F and primer12_Bf-infusion-R using the PCR product of the first step as a template.
  • PCR was performed using primer10_Bf-eno-Atsignal-F and primer12_Bf-infusion-R as the third step, using the PCR product of the second step as a template.
  • PCR was performed using primer11_Bf-infusion-F and primer12_Bf-infusion-R using the PCR product of the third step as a template as the fourth step.
  • the obtained PCR product contains an intron that encodes a sequence in which 16 amino acids (MYRLLSTFASVAAA) of the signal sequence portion of the full-length amino acid sequence of BfGLD (SEQ ID NO: 2) are replaced with the AtGLD signal sequence (SEQ ID NO: 10). It is a polynucleotide.
  • the sequence containing no intron of the sequence is shown as SEQ ID NO: 42 as the Atsig-16AA ( ⁇ ) BfGLD gene, and the amino acid sequence encoded by the sequence is shown in SEQ ID NO: 43.
  • PCR was performed using primer19_Bf-Aosig-short1-16AA-F and primer12_Bf-infusion-R using the plasmid extracted in (2) of Example 2 as a template.
  • PCR was performed using primer14_Bf-Aosig-short2-F and primer12_Bf-infusion-R using the PCR product of the first step as a template.
  • PCR was performed using primer17_Bf-eno-Aosignal-F and primer12_Bf-infusion-R as the third step, using the PCR product of the second step as a template.
  • PCR was performed using primer11_Bf-infusion-F and primer12_Bf-infusion-R using the PCR product of the third step as a template as the fourth step.
  • the obtained PCR product encodes a sequence in which 16 amino acids (MYRLLLSTAVASLAAA) in the signal sequence portion of the full-length amino acid sequence of BfGLD (SEQ ID NO: 2) are replaced with a predicted signal sequence (SEQ ID NO: 12) of AoGLD.
  • a polynucleotide comprising The sequence containing no intron of the sequence is shown as SEQ ID NO: 44 as the AosigS-16AA ( ⁇ ) BfGLD gene, and the amino acid sequence encoded by the sequence is shown in SEQ ID NO: 45.
  • PCR was performed using primer20_Bf-Atsignal1-19AA-F and primer12_Bf-infusion-R using the plasmid extracted in (4) of Example 2 as a template.
  • PCR was performed using primer9_Bf-Atsignal2-F and primer12_Bf-infusion-R using the PCR product of the first step as a template.
  • PCR was performed using primer10_Bf-eno-Atsignal-F and primer12_Bf-infusion-R as the third step, using the PCR product of the second step as a template.
  • PCR was performed using primer11_Bf-infusion-F and primer12_Bf-infusion-R using the PCR product of the third step as a template as the fourth step.
  • the obtained PCR product is a poly-containing intron that encodes a sequence in which the entire signal sequence 19 amino acids (MYRLLSTFASVALAASTD) of the full-length amino acid sequence of BfGLD (SEQ ID NO: 2) is replaced with the signal sequence of AtGLD (SEQ ID NO: 10). It is a nucleotide.
  • SEQ ID NO: 46 The sequence containing no intron of the sequence is shown as SEQ ID NO: 46 as the Atsig-19AA ( ⁇ ) BfGLD gene, and the amino acid sequence encoded by the sequence is shown in SEQ ID NO: 47.
  • PCR was performed using primer21_Bf-Aosig-short1-19AA-F and primer12_Bf-infusion-R using the plasmid extracted in (2) of Example 2 as a template.
  • PCR was performed using primer14_Bf-Aosig-short2-F and primer12_Bf-infusion-R using the PCR product of the first step as a template.
  • PCR was performed using primer17_Bf-eno-Aosignal-F and primer12_Bf-infusion-R as the third step, using the PCR product of the second step as a template.
  • PCR was performed using primer11_Bf-infusion-F and primer12_Bf-infusion-R using the PCR product of the third step as a template as the fourth step.
  • the obtained PCR product encodes a sequence in which the entire signal sequence 19 amino acids (MYRLLSTFASVALAASTAST) of the full-length amino acid sequence of BfGLD (SEQ ID NO: 2) is replaced with the expected signal sequence (SEQ ID NO: 12) of AoGLD.
  • a polynucleotide comprising. The sequence containing no intron of this sequence is shown as SEQ ID NO: 48 as the AosigS-19AA ( ⁇ ) BfGLD gene, and the amino acid sequence encoded by the sequence is shown in SEQ ID NO: 49.
  • Each of these expression vectors is introduced into E. coli strain JM109 for transformation, and the resulting transformants are cultured. From the collected cells, Illustra plasmid-prep MINI Flow Kit (manufactured by GE Healthcare) ), And each of the inserts was subjected to sequence analysis. Atsig-7AA ( ⁇ ) BfGLD gene, AosigS-7AA ( ⁇ ) BfGLD gene, AosigL-7AA ( -) BfGLD gene, Atsig-16AA (-) BfGLD gene, AosigS-16AA (-) BfGLD gene, Atsig-19AA (-) BfGLD gene and AosigS-19AA (-) BfGLD gene were confirmed.
  • the N-terminus of the enzyme sample derived from the Atsig-7AA ( ⁇ ) BfGLD gene was analyzed and found to be STLNY. That is, it was found that even when a part of the original signal sequence of the wild type was deleted and another signal sequence was added, the N-terminus of the enzyme was the same STLNY as that of the wild type.
  • Example 7 (Thermal stability) Sc (+) BfGLD is mixed with SPB (pH 5.0) kept at 40 ° C. or 45 ° C. so that the final concentration is Sc (+) BfGLD 6 U / mL, 100 mM sodium phosphate buffer (SPB), It was left in a hot water bath at 40 ° C. or 45 ° C. for 5 to 20 minutes. Enzyme heat-treated at each temperature for 5 minutes, 10 minutes, 15 minutes or 20 minutes was measured by the enzyme activity measurement method, and the residual activity of the enzyme heat-treated for each time when the activity of the enzyme without heat treatment was defined as 100%. Was calculated and shown in FIG.
  • the enzyme of the present application retained an activity of 80% or more after treatment at 40 ° C. for 20 minutes. On the other hand, it was found that the activity rapidly decreased by keeping the enzyme solution at 45 ° C. Therefore, it is important not to leave the enzyme solution at 45 ° C. for 5 minutes or longer in order to suppress enzyme deactivation in the sensor manufacturing process.

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Abstract

L'invention concerne une glucose déshydrogénase provenant de Botryotinia fuckeliana, un procédé de production de celle-ci, et une méthode de dosage, une composition de réactif et un biocapteur, utilisant chacun l'enzyme mentionnée ci-dessus. L'invention concerne un biocapteur pour le dosage du glucose sans être affecté par l'oxygène dissous, caractérisé en ce qu'il utilise une glucose déshydrogénase liée à une flavine qui comprend un glycopolypeptide (e) ou (f) : (e) un glycopolypeptide comprenant une séquence d'acides aminés représentée par SEQ ID No:5 ; et (f) un glycopolypeptide comprenant une séquence d'acides aminés qui est issue de la séquence d'acides aminés représentée par SEQ ID No:5 par substitution, délétion ou addition d'un à plusieurs acides aminés et ayant une activité glucose déshydrogénase.
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Publication number Priority date Publication date Assignee Title
US10077432B2 (en) 2014-03-21 2018-09-18 Ikeda Food Research Co., Ltd. Flavin-conjugated glucose dehydrogenase
US10961514B2 (en) 2016-01-14 2021-03-30 Ikeda Food Research Co., Ltd. Flavin-conjugated glucose dehydrogenase

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