WO2020003752A1 - Protéine ayant une activité fructosyl valyl histidine oxydase - Google Patents

Protéine ayant une activité fructosyl valyl histidine oxydase Download PDF

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WO2020003752A1
WO2020003752A1 PCT/JP2019/018491 JP2019018491W WO2020003752A1 WO 2020003752 A1 WO2020003752 A1 WO 2020003752A1 JP 2019018491 W JP2019018491 W JP 2019018491W WO 2020003752 A1 WO2020003752 A1 WO 2020003752A1
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protein
amino acid
present
glycated
acid sequence
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Japanese (ja)
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川井　淳
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Toyobo Co Ltd
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Toyobo Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

Definitions

  • the present invention relates to a glycated protein, particularly a fructosyl amino acid oxidase useful for measuring glycated hemoglobin, a method for measuring a glycated protein using the same, a glycated protein measurement kit, and a glycated protein measurement sensor. More specifically, the present invention relates to a fructosyl amino acid oxidase having improved stability to a quaternary ammonium salt, a glycated protein measurement method using the same, a glycated protein measurement kit, and a glycated protein measurement sensor.
  • HbA1c hemoglobin A1c
  • glycoalbumin which is a glycated protein contained in blood proteins, as a blood sugar control marker.
  • Glycated proteins are produced by the reaction between D-glucose present in blood and amino acid residues constituting blood proteins.
  • the major glycation sites in blood proteins are the ⁇ -amino group of lysine residues and the ⁇ -amino group of the amino-terminal amino acid of blood proteins.
  • glycated proteins can be measured at high throughput, which is useful in the field of clinical testing.
  • a glycated protein is hydrolyzed by a protease.
  • the resulting glycated amino acids such as fructosyl valine, fructosyl lysine and fructosyl valyl histidine are then oxidatively hydrolyzed by fructosyl valyl histidine oxidase.
  • hydrogen peroxide generated by the oxidase reaction is colorimetrically determined by a peroxidase-chromogen reaction system (see Patent Documents 1 to 11).
  • fructosyl valyl histidine oxidase which is the main reaction enzyme, is an important factor in the measurement of glycated proteins by the enzymatic assay.
  • an enzyme having high specificity for fructosyl valine is desired.
  • an enzyme acting on fructosyl valyl histidine is desired.
  • fructosyl valyl histidine (fructosyl valyl histidine oxidase) has been extracted and purified from several types of filamentous fungi or their genetically modified products. Also, a gene encoding this fructosyl valyl histidine oxidase has been isolated (see Patent Document 14, Non-patent Document 1, Non-patent Document 2).
  • a protease is used to hydrolyze glycated proteins as described above.
  • dimethyl sulfoxide as a stabilizing agent for suppressing a decrease in protease activity during storage of a reagent in a liquid state
  • Alcohol, calcium, salt, quaternary ammonium salts, quaternary ammonium salt type cationic surfactants and the like are mentioned (Patent Document 16).
  • Leuco dyes have the advantage of extremely high measurement sensitivity and are less susceptible to the effects of hemoclobin, bilirubin, and the like in the measurement sample because the absorption maximum is on the longer wavelength side as compared with conventional Trinder reagents.
  • the leuco dye does not have sufficient storage stability in a solution, and gradually becomes colored during storage.
  • Patent Document 17 a method has been disclosed in which a quaternary ammonium salt-type cationic surfactant is used as a stabilizer for a leuco dye as a stabilizer.
  • Hirokawa @ K. , ⁇ Gomi ⁇ K. , ⁇ And ⁇ Kajiyama @ N. Molecular cloning and expression of novel fructosyl peptide oxidases and their application for the measurement of lgrated , @Biochem. ⁇ Biophys. Res. ⁇ Commun. , $ 2003, $ 311 (1), $ 104-111.
  • fructosyl valyl histidine oxidase has poor stability in the presence of a quaternary ammonium salt, and further improvement in stability when used in a diagnostic reagent is desired.
  • an object of the present invention is to provide a fructosyl bacterium having excellent stability against quaternary ammonium salts, which is useful for measuring glycated proteins, particularly glycated hemoglobin.
  • the present inventors have conducted intensive studies to achieve the above object, and as a result, a protein having fructosyl valyl histidine oxidase activity useful for measuring fructosyl amino acid, which is more stable to quaternary ammonium salts than wild type.
  • the present inventors have confirmed that it is possible to measure fructosyl amino acid by allowing this mutant protein to act on a fructosyl amino acid-containing specimen and quantifying the generated hydrogen peroxide by a peroxidase reaction.
  • the present invention has been completed. That is, the present invention is exemplified as follows.
  • a protein having fructosyl valyl histidine oxidase activity according to any one of the following (a) to (c): (A) In the amino acid sequence represented by SEQ ID NO: 1, glutamic acid at at least one site selected from the group consisting of the 101st, 131st, 169th, 245th, and 360th positions is replaced with another amino acid. In the protein (b) consisting of the amino acid sequence (a), one or several amino acids are deleted, substituted or added at positions other than the 101st, 131st, 169th, 245th and 360th positions.
  • Item 1 The protein according to Item 1, wherein the amino acid sequence before amino acid substitution in (c) has 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1.
  • Item 3. The protein according to Item 1 or 2, wherein the substituted amino acid is any one selected from the group consisting of lysine, arginine and histidine.
  • Item 5. A polynucleotide encoding the protein of any one of Items 1 to 4.
  • Item 6. Item 6.
  • Item 7. Item 7.
  • Item 10. A method for producing a protein having fructosyl valyl histidine oxidase activity comprising the step of culturing the transformant of Item 7 to produce a protein having fructosyl valyl histidine oxidase activity, and collecting the protein.
  • a method for measuring a glycated protein comprising a step of allowing the protein of any one of Items 1 to 4 to act on a glycated amine.
  • Item 10. Item 7. A glycated protein measurement kit comprising the protein according to any one of Items 1 to 4.
  • Item 11. Item 11.
  • Item 4. A glycated protein measurement sensor for measuring an electric current generated by a reaction of a protein, using an electrode to which the protein according to any one of Items 1 to 4 is immobilized.
  • the protein according to the present invention is obtained by substituting at least one of the amino acids corresponding to glutamic acid at positions 101, 131, 169, 245 and 360 in the amino acid sequence represented by SEQ ID NO: 1 and its analogous sequence. And a protein having a substitution oxidase activity of a fructosyl amino acid.
  • the protein is particularly effective in providing a protein having fructosyl valyl histidine oxidase activity having excellent stability to quaternary ammonium salts.
  • the protein according to the present invention is a protein having fructosyl valyl histidine oxidase activity described in any of the following (a) to (c).
  • amino acid sequence (a) consisting of a sequence one or several amino acids are deleted, substituted or added at a site other than the 101st, 131st, 169th, 245th and 360th positions
  • At least one selected from the group consisting of the 169th, 245th and 360th Protein is also at least one of glutamic acid corresponding to the site of any one
  • the protein according to the present invention exhibits excellent stability even in the presence of a quaternary ammonium salt, as compared with the protein before modification, by constituting the modified amino acid sequence as described above. This is because by replacing the negatively charged amino acid residue located on the enzyme surface with a positively charged amino acid residue, the electric repulsion against quaternary ammonium salts was increased, and the interference with the enzyme was reduced. It is presumed to be the effect of this.
  • the sequence having an identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 1 may have a different length from the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence identity can be calculated by using a commercially available or free software such as GENETYX-WIN (manufactured by Genetics) to calculate the proportion (%) of matching sequences by homology search of the two types of sequences. it can.
  • amino acid residue substitution sites are 101, 131, 169, and 245 from the amino terminal of the amino acid sequence shown in SEQ ID NO: 1 as long as they have fructosyl valyl histidine oxidase activity.
  • the position is not particularly limited as long as it is at least one site of the glutamic acid at the 3rd and 360th positions, and may be a single amino acid at the 5 positions or a combination of these multiple positions.
  • the amino acid to be substituted is not particularly limited as long as it has fructosyl valyl histidine oxidase activity, but is preferably lysine, arginine, histidine, and more preferably lysine. No. If the homology is 85% or more with the amino acid sequence shown in SEQ ID NO: 1, sites corresponding to these residues can be determined using, for example, GENETYX-WIN described above.
  • the present invention also relates to the amino acid sequence of the protein according to the present invention as described above, which comprises an amino acid sequence in which one or several amino acids have been deleted, substituted and / or added, and has an activity of fructosyl valyl histidine oxidase.
  • proteins having the formula: The site where one or several amino acids are deleted, substituted and / or added is a site other than amino acids 101, 131, 169, 245 and 360 from the amino terminus, and If the protein after substitution and / or addition has fructosyl valyl histidine oxidase activity, it may be an amino acid at any position in the amino acid sequence.
  • “one or several amino acids” refers specifically to the number of amino acids in a range of 10 or less.
  • fructosyl valyl histidine oxidase activity in the present invention is measured by the method described in the section of “Activity measurement method” in Examples described later.
  • “having fructosyl valyl histidine oxidase activity” means preferably having an activity of 0.1 U / mg-protein or more, more preferably 1.0 U / mg-protein. It means having the above activity.
  • the protein according to the present invention may be produced, for example, using a genetic recombination technique using a polynucleotide and a vector described later, or may be chemically synthesized using an amino acid synthesizer or the like.
  • various recombinant protein expression systems suitably used include, but are not limited to, for example, an E. coli expression system, an insect cell expression system, a mammalian cell expression system, and a cell-free expression system. The method for producing the protein or the like of the present invention will be described later.
  • the protein according to the present invention may be, for example, one having undergone intermolecular and / or intramolecular cross-linking (for example, disulfide bond), or chemically modified (for example, addition of sugar chain, phosphorylation or other functional group). But not limited thereto, such as those to which a label (for example, a histidine tag or the like) is added, or those to which a fusion protein (for example, streptavidin, cytochrome or GFP) is added.
  • the protein and the like according to the present invention may also include a chimeric protein constituted by combining fragments of several kinds of proteins, as long as fructosyl valyl histidine oxidase activity is substantially maintained.
  • the fructosyl valyl histidine oxidase agent may be composed of the protein according to the present invention, serum proteins, organic acids, and excipients such as dextran.
  • the fructosyl valyl histidine oxidase agent may contain a known component as a component of the enzyme agent.
  • the protein according to the present invention is characterized in that the stability in the presence of a quaternary ammonium salt is improved by amino acid substitution as compared to before the amino acid substitution. As a result, long-term storage stability when the protein is used in a diagnostic reagent is improved.
  • the quaternary ammonium salt in this case for example, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, Hexadecyltrimethylammonium, octadecyltrimethylammonium bromide, di (dodecyl) dimethylammonium chloride, di (tetradecyl) dimethylammonium chloride, di (hexadecyl) dimethylammonium chloride, di (octadecyl) dimethylammonium chloride, di (dodecyl) dimethylbromide Ammonium, di (tetradecyl) dimethylammonium bromide, di (hexadecyl)
  • the stability in the presence of a quaternary ammonium salt is defined as 0.01 to 1% (W / V), preferably 0.04 to 0.1% (W / V) in a 1 U / ml protein solution. ) At 25 ° C., preferably 30% or more, more preferably 40% or more, when the treatment is carried out at 30 ° C. for 5 to 10 minutes under the condition that a quaternary ammonium salt coexists. % Or more.
  • polynucleotide is characterized by comprising a base sequence encoding the protein according to the present invention.
  • the base sequence of SEQ ID NO: 3 is exemplified.
  • the polynucleotide may exist in the form of DNA (eg, cDNA or genomic DNA) or RNA (eg, mRNA).
  • DNA or RNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand).
  • the polynucleotide according to the present invention may be chemically synthesized, and the codon usage (codon usage; Codon usage) may be changed so that the expression of the encoded protein is improved.
  • a method for modifying the polynucleotide according to the present invention a commonly used polynucleotide modification method is used. That is, a polynucleotide having the genetic information of the recombinant protein may be prepared by substituting, deleting, and / or adding a specific base of the polynucleotide having the genetic information of the protein.
  • a specific method for converting the base of the polynucleotide for example, a commercially available kit (Transformer Site-Directed Mutagenesis Kit; manufactured by Clontech, QuikChange Site Directed Mutagensis Kit; polymerase chain reaction, PCR method, etc.) Use. These methods are known to those skilled in the art.
  • the polynucleotide according to the present invention may be substituted with a chemically synthesized base.
  • the site where the polynucleotide according to the present invention is substituted is not particularly limited as long as the protein expressed from the base sequence after substitution has suitable properties.
  • the polynucleotide according to the present invention may be composed of only the polynucleotide encoding the protein according to the present invention, or may have another base sequence added thereto.
  • the base sequence to be added include, but are not limited to, a label (for example, a histidine tag, a Myc tag and a FLAG tag), a fusion protein (for example, GST and MBP), or a signal sequence (for example, an endoplasmic reticulum translocation signal sequence and secretion).
  • promoter sequences eg, yeast-derived promoter sequences, phage-derived promoter sequences, E. coli-derived promoter sequences, and the like.
  • the site to which these nucleotide sequences are added is not particularly limited as long as the desired function of the translated protein is maintained, but it should be a site corresponding to the N-terminus or C-terminus of the translated protein. Is preferred.
  • polynucleotide according to the present invention hybridizes under stringent conditions with the polynucleotide encoding the protein according to the present invention described above, or a polynucleotide consisting of a nucleotide sequence complementary thereto, and has the sequence of SEQ ID NO: 1.
  • amino acid sequence in which at least one of the amino acids corresponding to positions 101, 131, 169, 245 and 360 from the amino terminus of the amino acid sequence shown has been substituted with the amino acid sequence shown in SEQ ID NO: 1
  • a polynucleotide encoding a protein having fructosyl valyl histidine oxidase activity consisting of Polynucleotides consisting of these sequences can also be obtained by commonly used polynucleotide modification methods.
  • stringent conditions refers to conditions under which the nucleic acids hybridize at a temperature ranging from the Tm value of nucleic acids having high homology, for example, perfectly matched hybrids, to 15 ° C., preferably 10 ° C. lower. . As a specific example, it refers to conditions for hybridization in a general hybridization buffer at 68 ° C. for 20 hours.
  • the vector according to the present invention contains the above-described polynucleotide according to the present invention.
  • Other configurations are not particularly limited as long as they include the polynucleotide according to the present invention.
  • a base vector constituting the vector according to the present invention a vector suitable for a host cell can be appropriately selected.
  • pBluescript registered trademark
  • pUC18 can be used.
  • the host microorganism or host cell into which the vector is introduced for example, yeast, Escherichia coli (for example, Escherichia coli (Escherichia coli) W3110, Escherichia coli C600, Escherichia coli JM109, Escherichia coli DH5 ⁇ ), insects Cells and mammalian cells can be used.
  • Escherichia coli for example, Escherichia coli (Escherichia coli) W3110, Escherichia coli C600, Escherichia coli JM109, Escherichia coli DH5 ⁇
  • insects Cells and mammalian cells can be used.
  • the method for introducing the vector according to the present invention into the above-described host microorganism or host cell is not particularly limited.
  • the method when introducing a vector into a microorganism belonging to the genus Escherichia as a host microorganism, the method may be carried out in the presence of calcium ions. And a method using an electroporation method.
  • gene transfer may be performed using commercially available competent cells (for example, competent high JM109, competent high DH5 ⁇ ; manufactured by Toyobo).
  • the vector according to the present invention after separating and purifying the polynucleotide according to the present invention, a fragment of the polynucleotide cut using a restriction enzyme or the like, and the base vector is cut with a restriction enzyme.
  • the resulting linear polynucleotide can be constructed by binding and closing. In closing the binding, DNA ligase or the like can be used depending on the properties of the vector and the polynucleotide.
  • a transformant containing the polynucleotide of the present invention can be obtained by screening using the markers of the vector and the expression of enzyme activity as indices. Therefore, the vector according to the present invention preferably contains a marker gene such as a drug resistance gene.
  • the present invention also encompasses a transformant transformed with the above-described vector of the present invention, that is, a transformant containing the vector of the present invention.
  • Host cells transformed with the vector according to the present invention are not particularly limited, and include yeast, Escherichia coli, insect cells, mammalian cells, and the like.
  • the method for producing a protein according to the present invention is characterized by including the step of culturing the above-described transformant according to the present invention (referred to as “culturing step”).
  • the protein production method according to the present invention may include, in addition to the culturing step, other steps that can be included in the production of a protein using a transformant. Other steps include, for example, a recovery step of recovering the protein produced by the transformant after the culture step, and a purification step of purifying the protein.
  • (3-1) Culture step In the culture step, a large amount of recombinant protein can be stably produced by culturing the transformant according to the present invention in a nutrient medium.
  • the form of culture of the transformant may be selected in consideration of the nutritional and physiological properties of the host, and in most cases, liquid culture is used. Industrially, it is advantageous to carry out aeration and stirring culture.
  • the carbon source may be any assimilable carbon compound, for example, glucose, sucrose, lactose, maltose, lactose, molasses, pyruvic acid and the like.
  • the nitrogen source may be any nitrogen compound that can be used, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract.
  • salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron and manganese zinc, amino acids, vitamins and the like may be added to the medium as needed.
  • the culture temperature of the transformant according to the present invention can be appropriately changed as long as the transformant can produce the protein according to the present invention.
  • Escherichia coli when used as a host, it is preferably used. It is about 20 to 42 ° C.
  • the culturing time may be completed at an appropriate time when the protein of the present invention reaches the maximum yield, and is usually about 6 to 48 hours.
  • the pH of the medium can be appropriately changed within a range in which the transformant can suitably grow and the protein according to the present invention can be produced, but is preferably in the range of about 6.0 to 9.0.
  • the culture contains the protein of the present invention. Therefore, the culture can be used as it is as the protein of the present invention. At this time, the culture solution and the transformant may be separated by, for example, filtration and centrifugation.
  • the transformant When the protein according to the present invention is present in a transformant, the transformant is collected from a culture obtained by culturing the transformant by means such as filtration and centrifugation, and the collected transformant is collected. May be destroyed by a mechanical method or an enzymatic method such as lysozyme, and then the target protein may be recovered. If necessary, a protein according to the present invention may be solubilized by adding a chelating agent (eg, EDTA) and a surfactant (eg, Triton-X100), and may be separated and collected as an aqueous solution.
  • a chelating agent eg, EDTA
  • a surfactant eg, Triton-X100
  • the purification step is a step of purifying the protein obtained in the recovery step.
  • a specific method of the purification step is not particularly limited, for example, a solution containing the protein according to the present invention is concentrated under reduced pressure, membrane concentrated, salted out (for example, using ammonium sulfate and sodium sulfate), Alternatively, it may be subjected to a fractional precipitation method using a hydrophilic organic solvent (eg, methanol, ethanol, acetone, etc.). By these operations, the target protein of the present invention can be precipitated and purified.
  • a hydrophilic organic solvent eg, methanol, ethanol, acetone, etc.
  • purification may be performed by heat treatment, isoelectric point treatment, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, reverse phase chromatography, or a combination thereof.
  • the purified enzyme containing the target protein obtained by using these techniques is purified to such an extent that it shows a single band in electrophoresis (SDS-PAGE).
  • the above purified enzyme can be powdered by, for example, freeze-drying, vacuum drying, spray drying and the like and distributed.
  • the purified enzyme can be used in a state of being appropriately dissolved in a buffer depending on the use.
  • a buffer for example, a borate buffer, a phosphate buffer, a Tris-HCl buffer, a GOOD buffer, and the like may be suitably selected according to the properties of the target protein and / or experimental conditions or environment. .
  • it can be stabilized by adding amino acids (for example, glutamic acid, glutamine and lysine, etc.), serum albumin and the like to the purified enzyme.
  • the protein according to the present invention can be used for a method for measuring fructosyl amino acid. Further, the method for measuring fructosyl amino acid using the protein according to the present invention can be applied to the measurement of glycated proteins such as glycated hemoglobin.
  • glycated proteins such as glycated hemoglobin.
  • the measurement method of the present invention is characterized by causing the protein of the present invention to act on at least glycated amine.
  • the present invention is not limited thereto.
  • One embodiment of the measuring method according to the present invention (1) reacting a sample with a protease to degrade glycated protein in the sample and prepare a glycated amine derived from the glycated protein in the sample (referred to as “first step” for convenience); (2) a step of allowing the protein according to the present invention to act on a glycated amine derived from a glycated protein in the sample obtained by the step (1) (for convenience, referred to as a “second step”); (3) A method for measuring a glycated protein comprising a step of measuring the amount of hydrogen peroxide generated in the step (2) or the amount of consumed oxygen (referred to as “third step” for convenience). .
  • One specific example of this embodiment is an enzymatic method.
  • a glycated protein in a sample is fragmented to a level of amino acids or peptides using an enzyme (for example, a protease) (first step).
  • an enzyme for example, a protease
  • fructosyl valyl histidine oxidase is added to the resulting glycated amino acid and / or glycated peptide, and hydrogen peroxide is generated by an oxidation-reduction reaction (second step).
  • Peroxidase (POD) and a reducing agent that develops color by oxidation are added to this sample, and a redox reaction is caused between hydrogen peroxide and the reducing agent using POD as a catalyst (third step).
  • the amount of hydrogen peroxide can be measured by coloring the reducing agent by an oxidation-reduction reaction and measuring the intensity of the coloring (third step).
  • the first step is a step of decomposing a glycated protein in a sample to prepare a glycated amine derived from the glycated protein in the sample.
  • glycated protein means a protein in which a sugar is bound (glycated) to a part or all of the amino acid residues constituting the protein.
  • the glycated protein is not particularly limited, and includes, for example, a protein in which the ⁇ -amino group at the amino terminal of the protein is glycated (eg, HbA1c).
  • HbA1c exemplified above is widely used as an index for clinical diagnosis such as diagnosis of diabetes.
  • the protease used in the first step is not particularly limited as long as it can degrade a glycated protein contained in a sample into a glycated amino acid or a glycated peptide.
  • proteases derived from animals, plants, bacteria such as Bacillus, fungi such as Aspergillus, and yeast are preferred.
  • Preferred examples of proteases derived from microorganisms of the genus Bacillus include subtilisin and metalloprotease.
  • the ⁇ sample '' used in the present measurement method is not particularly limited as long as it is a target for which the presence or absence or concentration of a glycated protein is to be detected, and for example, besides whole blood, plasma, serum and blood cells, etc. And biological samples such as urine and cerebrospinal fluid (that is, samples collected from a living body), and samples such as drinking water such as juice and foods such as soy sauce and sauce. Since the method of the present invention can be applied to the diagnosis of diabetes, it is particularly useful when measuring a whole blood sample and a blood cell sample among the above.
  • hemolyzed sample when measuring glycated hemoglobin in erythrocytes, whole blood is directly lysed or erythrocytes separated from whole blood are lysed, and this hemolyzed sample is used as a sample for measurement. It may be.
  • Specific conditions for reacting the sample with the protease are not particularly limited as long as the desired saccharified amine can be prepared, and may vary depending on the concentration and type of the sample and the concentration and type of the protease. Appropriate conditions can be appropriately examined and adopted.
  • the concentration of protease that can be suitably used in the measurement method of the present invention is, for example, 0.1 U to 1 MU / ml, more preferably 1 U to 500 KU / ml, and most preferably 5 U to 100 KU / ml.
  • the concentration of the protease is not limited, and can be suitably determined by the experimenter or the user depending on the reaction conditions, the type and state of the sample, the procedure of the experimenter, the type of the reagent to be used, and the like.
  • the “glycated amine” on which the protein according to the present invention acts includes glycated amino acids and glycated peptides derived from glycated proteins contained in the sample.
  • the length of the glycated peptide is not particularly limited, but the length at which the protein of the present invention can act includes, for example, those having about 2 to 6 amino acid residues.
  • the second step is a step of allowing the protein according to the present invention to act on a glycated amine derived from a glycated protein in the sample obtained in the first step.
  • the fructosyl valyl histidine oxidase activity of the protein according to the present invention is not particularly limited, but the higher the fructosyl valyl histidine oxidase activity, the higher the sensitivity with which glycated amines can be detected.
  • the histidine oxidase protein is preferable because the amount of use can be reduced.
  • the concentration of the fructosyl valyl histidine oxidase protein used in the measurement method of the present invention is, for example, 0.1 to 500 U / ml, preferably 0.5 to 200 U / ml, and most preferably 1.0 to 200 U / ml. 100100 U / ml.
  • the concentration of the above fructosyl valyl histidine oxidase is not particularly limited, and can be appropriately determined according to the reaction conditions, the type and state of the sample, the procedure of the experimenter, the type of the reagent to be used, and the like. .
  • the third step is a step of measuring the amount of hydrogen peroxide generated in the second step or the amount of consumed oxygen.
  • the specific method of the third step is not particularly limited as long as the method can measure the amount of hydrogen peroxide or the amount of oxygen. Therefore, known methods can be appropriately applied.
  • the amount of hydrogen peroxide is measured by adding POD and a reducing agent that develops color by oxidation to the sample obtained in the second step, and measuring the coloring intensity of the reducing agent. .
  • suitable PODs include those derived from horseradish and microorganisms. Further, the preferred working concentration of POD is 0.01 to 100 units / mL.
  • a suitable method for measuring hydrogen peroxide in the third step is to use a coupler (such as 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazone (MBTH)) in the presence of POD.
  • a coupler such as 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazone (MBTH)
  • MBTH 3-methyl-2-benzothiazolinone hydrazone
  • Trinder reagent that generates a dye by oxidative condensation reaction of a phenol-based, aniline-based or toluidine-based chromogen, and a leuco dye that directly oxidizes and colors in the presence of POD.
  • Trinder reagent for example, phenol and its derivatives can be used.
  • Couplers that can be suitably used in the third step include 4-aminoantipyrine, aminoantipyrine derivatives, vanillin diamine sulfonic acid, methylbenzthiazolinone hydrazone (MBTH), and sulfonated methylbenzthiazolinone hydrazone (SMBTH). No.
  • the leuco dye that can be suitably used in the third step is not particularly limited, but for example, a triphenylmethane derivative, a phenothiazine derivative, a diphenylamine derivative and the like can be used.
  • a measuring method using various sensor systems is generally known to those skilled in the art in addition to a coloring method using POD or the like (not limited to, for example, 2001-204494).
  • electrodes used for various sensor systems include an oxygen electrode, a carbon electrode, a gold electrode, and a platinum electrode.
  • these electrodes on which an enzyme is immobilized are used as working electrodes, and a buffer solution under conditions suitable for the present invention is used together with a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / Cl electrode).
  • a constant voltage is applied to the working electrode while being kept at a constant temperature by inserting it into the working electrode, and a sample is further added to measure an increase in current caused by hydrogen peroxide resulting from the enzymatic reaction.
  • a method of measuring with an amperometric system using a carbon electrode, a gold electrode, a platinum electrode, or the like there is a system using an immobilized electron mediator.
  • these electrodes in which an enzyme and an electron mediator such as potassium ferricyanide, ferrocene, osmium derivative, and phenazine methosulfate are adsorbed or immobilized on a polymer matrix by a covalent bonding method as a working electrode are used as a counter electrode (for example, a platinum electrode).
  • a reference electrode eg, an Ag / AgCl electrode, etc.
  • a reference electrode eg, an Ag / AgCl electrode, etc.
  • a constant voltage is applied to the working electrode, the sample is added, and the increase in current due to hydrogen peroxide resulting from the enzymatic reaction can be measured.
  • the amount of saccharified amine can also be measured by measuring the amount of oxygen consumed in the third step (for example, but not limited to, refer to JP-A-2001-204494). Specifically, using an oxygen electrode, oxygen is immobilized on the electrode surface, and inserted into a buffer solution under conditions suitable for the present invention, and maintained at a constant temperature. The sample is added here, and the decrease value of the current is measured.
  • these electrodes on which an enzyme is immobilized are used as a working electrode, and a counter electrode (for example, a platinum electrode or the like) and a reference electrode (for example, , Ag / AgCl electrodes, etc.) and the current increase including the mediator is measured.
  • a counter electrode for example, a platinum electrode or the like
  • a reference electrode for example, Ag / AgCl electrodes, etc.
  • the mediator for example, compounds such as potassium ferricyanide, ferrocene, osmium derivatives, and phenazine methosulfate can be used.
  • an inactive protein may be added to the system for performing each step in order to increase the stability of the protein of the present invention.
  • Inactive proteins include serum albumins, globulins and fibrous proteins.
  • a preferred protein is bovine serum albumin, a preferred concentration is 0.05-1% (W / V).
  • Preferred inactive proteins are those that do not contain protease impurities that cause enzymatic degradation.
  • Glycated protein measurement kit A glycated protein measurement kit according to the present invention (hereinafter referred to as “kit of the present invention”) is characterized by containing at least the protein according to the present invention.
  • the form of the protein according to the present invention provided in the kit of the present invention is not particularly limited.
  • a form such as an aqueous solution, a suspension, or a lyophilized powder can be adopted.
  • the lyophilized powder can be prepared according to a conventional method.
  • the method of compounding the above additives is not particularly limited.
  • a method of blending an additive with a buffer containing a protein according to the present invention a method of blending a protein according to the present invention with a buffer containing an additive, or a buffer comprising a protein and a stabilizer according to the present invention in a buffer A method of mixing them at the same time can be used.
  • the kit of the present invention is exemplified by an embodiment constituted by a reagent composition comprising at least the protein, peroxidase and chromogen according to the present invention.
  • peroxidase reaction for detecting hydrogen peroxide derived from oxidation of fructosyl amino acid. Therefore, peroxidase and a chromogen (hydrogen peroxide coloring reagent) are preferably used in the reagent composition.
  • the chromogen (hydrogen peroxide coloring reagent) used in the present invention is preferably stable in solution and has low bilirubin interference.
  • the chromogen (hydrogen peroxide coloring reagent) that can be suitably used in the present invention include, for example, 4-aminoantipyrine or 3-methyl-2-benzothiazoline hydrazone (MBTH) and phenol or a derivative thereof, or aniline or a derivative thereof.
  • a reagent comprising a combination of The chromogen (hydrogen peroxide coloring reagent) used in the present invention may be a benzidine, a leuco dye, a 4-aminoantipyrine, a phenol, a naphthol, or an aniline derivative.
  • the peroxidase preferably used in the present invention is preferably a horseradish peroxidase. This peroxidase is commercially available in high purity and at low cost.
  • the enzyme concentration must be high enough for a quick and complete reaction, preferably between 1,000 and 50,000 U / L.
  • the reagent composition may contain a buffer (eg, a borate buffer, a phosphate buffer, a Tris-HCl buffer, a GOOD buffer, etc.) in addition to the above components.
  • the reagent composition includes chelating reagents (for example, EDTA and O-dianisidine) for capturing ions that interfere with the enzymatic reaction, ascorbate oxidase for eliminating ascorbic acid, which is an interfering substance for the determination of hydrogen peroxide, and various other reagents.
  • Surfactants eg, Triton X-100 and NP-40
  • various antibacterial and preservatives eg, streptomycin and sodium azide
  • These reagents may be a single reagent or a combination of two or more reagents.
  • the buffer is not particularly limited, but any buffer having a sufficient buffer capacity in a pH range of 6 to 8.5 can be used.
  • buffers include phosphate, tris, bis-trispropane, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 2-morpholinoethanesulfonic acid monohydrate ( (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (piperazine-1,4-bis (2-ethanesulfonic acid)) (PIPES), 2- [4- (2-hydroxyethyl) -1- Piperazinyl] ethanesulfonic acid (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid) (HEPES), and 3- [N-tris (hydroxymethyl) methylamino] -2-hydroxypropanesulfonic acid ( TAPSO).
  • preferred buffers are MES and PIPES.
  • the kit of the present invention includes, for example, fructosyl valyl histidine oxidase, a buffer, a protease, a POD, a coloring reagent, a chelating reagent that captures ions that interfere with the enzymatic reaction, and ascorbic acid, a substance that interferes with the determination of hydrogen peroxide.
  • Ascorbate oxidase surfactants, stabilizers, excipients, antibacterial agents, preservatives, well plates, fluorescent scanners, automatic analyzers, etc., for eliminating lipase.
  • the kit of the present invention can be used for the method for measuring fructosyl amino acid and the measurement of glycated protein according to the present invention, and can be particularly suitably used for measuring glycated hemoglobin.
  • the glycated protein measurement sensor according to the present invention is a sensor used for detecting a glycated protein, and includes at least the protein according to the present invention. I have.
  • the sensor of the present invention is used for carrying out the measuring method of the present invention. In particular, it can be suitably used for measurement of glycated hemoglobin. Therefore, the sensor of the present invention may be constituted by an article used for performing the measuring method of the present invention.
  • the description of the buffering agent and the like in the section of the measuring method of the present invention and the kit of the present invention can be referred to.
  • the sensor of the present invention includes an embodiment in which the protein of the present invention is immobilized on a support.
  • the support is not particularly limited as long as it can immobilize the protein according to the present invention, and a suitable shape and material may be used according to the properties of the protein.
  • the shape of the support is not particularly limited as long as it has a sufficient area on which the protein can be immobilized, and examples thereof include a substrate, beads, and a membrane.
  • the material of the support include inorganic materials, natural polymers and synthetic polymers.
  • Table 1 shows the composition of the activity measurement reagent used in the following examples.
  • the reagents used in the examples were purchased from Nacalai Tesque, unless otherwise specified.
  • the enzymatic activity for each substrate was determined by measuring the increase in the absorbance of the dye produced by the peroxidase reaction following the hydrogen peroxide produced by the enzymatic reaction. First, after preliminarily heating 3 ml of the activity measurement reagent at 37 ° C. for 5 minutes, 0.1 ml of an enzyme solution previously diluted with an enzyme diluent (50 mM potassium phosphate buffer (pH 7.5)) was added to the activity measurement reagent. Start the reaction. The reaction is performed at 37 ° C. for 5 minutes, and the change in absorbance at 500 nm is measured ( ⁇ ODtest / min).
  • Activity value (U / ml) ⁇ (( ⁇ ODtest / min) ⁇ ( ⁇ ODblank / min)) ⁇ 3.1 ml ⁇ dilution factor ⁇ / (13 ⁇ 1.0 cm ⁇ 0.1 ml) 3.1 ml: total solution volume 13: mmol extinction coefficient 1.0 cm: cell optical path length 0.1 ml: enzyme sample solution volume
  • Example 1 Subcloning of Fructosyl valyl histidine oxidase gene from Pheosperia nodrum> Large-scale expression in Escherichia coli of a plasmid containing the gene for fructosyl valyl histidine oxidase derived from Pheosperia nodrum having the nucleotide sequence shown in SEQ ID NO: 2 was attempted. The full-length cDNA region (from the 1st to the 1311th base of the nucleotide sequence shown in SEQ ID NO: 2) excluding the termination codon of the fructosyl valyl histidine oxidase gene derived from Pheospheria nodrum was amplified by PCR.
  • an NdeI cleavage site was inserted at the N-terminal side of the amino acid sequence using primer P5 (5′-GGAATTCCATATGGCGCCCTCCAGAGCAAAACACCAGTGTCCATT-3 ′) shown in SEQ ID NO: 4 (“CATATG” in this nucleotide sequence is an NdeI recognition site).
  • an XhoI cleavage site was introduced at the C-terminal side of the amino acid sequence using primer P6 (5′-CCGCTCGAGCAAGTTCGCCTCGGCTTATCATGATTCCAACC-3 ′) shown in SEQ ID NO: 5 (“CTCGAG” in this nucleotide sequence is an XhoI recognition site).
  • This DNA fragment was subcloned into the NdeI-XhoI site of the pET-23b vector (Novagen) so as to be in the forward direction with the T7 promoter.
  • the prepared plasmid was designated as pIE353 as a plasmid for expressing fructosyl valyl histidine oxidase.
  • Example 2 Construction, purification, and enzyme assay of a protein having fructosyl valyl histidine oxidase activity by modifying the function of the fructosyl valyl histidine oxidase gene derived from Pheosperia nodrum>
  • a mutation treatment operation was performed according to a specified protocol using KOD-Plus Site-Directed Mutagenesis Kit (manufactured by Toyobo) to determine the nucleotide sequence.
  • the nucleotide sequence encoding glutamic acid (E) at positions 101, 131, 169, 245 and 360 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with a nucleotide sequence encoding lysine (K).
  • Recombinant plasmids pIE353-E101K, pIE353-E131K, pIE353-E169K, pIE353-E245K, and pIE353-E360K
  • the transformant was cultured, and the resulting crude enzyme solution was purified to obtain purified enzyme preparations (IE353, IE353-E101K, IE353-E131K, IE353-E169K, IE353-E245K, IE353-E360K).
  • the fructosyl valyl histidine oxidase activity of the crude enzyme solution measured using the fructosyl valyl histidine-containing measuring reagent was 9.9 to 24.6 U / ml.
  • IE353-E101K, IE353-E131K, IE353-E169K, IE353-E245K, and IE353-E360K have higher stability to quaternary ammonium salts than the wild-type FAOD protein (IE353). Has improved.
  • the present invention it is possible to provide fructosyl valyl histidine oxidase having high stability to a quaternary ammonium salt useful for measuring fructosyl amino acid, and therefore, a method for measuring fructosyl amino acid using fructosyl valyl histidine oxidase And a fructosyl amino acid measurement reagent composition and the like. Therefore, the present invention can be widely used in the life science field, including the field of clinical testing based on preventive medicine, the field of diagnostic medicine, the field of pharmaceuticals, and the field of health medicine.

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Abstract

L'invention concerne une fructosyl-acide aminé oxydase ayant une stabilité améliorée pour un sel d'ammonium quaternaire et un procédé de mesure de protéine glyquée l'utilisant. La présente invention concerne une protéine ayant une activité fructosyl valyl histidine oxydase selon l'un quelconque de (a) à (c) ci-dessous : (a) une protéine comprenant, dans la séquence d'acides aminés présentée dans SEQ ID NO : 1, une séquence d'acides aminés dans laquelle l'acide glutamique au niveau d'au moins un site sélectionné dans un groupe constitué des 101ème, 131ème, 169ème, 245ème, et 360ème positions est substitué par un autre acide aminé ; (b) une protéine comprenant, dans la séquence d'acides aminés (I), une séquence d'acides aminés dans laquelle un ou plusieurs acides aminés sont supprimés, substitués ou ajoutés au niveau de sites autres que les 101ème, 131ème, 169ème, 245ème, et 360ème positions ; et (c) une protéine comprenant, dans la séquence d'acides aminés ayant au moins 85,0 % d'identité avec la séquence d'acides aminés représentée dans SEQ ID NO : 1, une séquence d'acides aminés dans laquelle l'acide glutamique correspondant à au moins un site sélectionné dans un groupe constitué des 101ème, 131ème, 169ème, 245ème, et 360ème positions dans SEQ ID NO : 1 est substitué par un autre acide aminé.
PCT/JP2019/018491 2018-06-27 2019-05-09 Protéine ayant une activité fructosyl valyl histidine oxydase Ceased WO2020003752A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010041419A1 (fr) * 2008-10-10 2010-04-15 東洋紡績株式会社 Nouvelle protéine ayant une activité de fructosyl-valyl-histidine oxydase et produit modifié de celle-ci et utilisation de la protéine ou du produit modifié
WO2015020200A1 (fr) * 2013-08-09 2015-02-12 キッコーマン株式会社 AMADORIASE MODIFIÉE ET SON PROCÉDÉ DE PRODUCTION, AGENT PERMETTANT D'AMÉLIORER LA RÉSISTANCE AUX TENSIOACTIFS DE L'AMADORIASE ET COMPOSITION PERMETTANT DE MESURER L'HbA1c À L'AIDE DE CELLE-CI
WO2016063984A1 (fr) * 2014-10-24 2016-04-28 キッコーマン株式会社 Amadoriase ayant une activité de déshydrogénase améliorée

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010041419A1 (fr) * 2008-10-10 2010-04-15 東洋紡績株式会社 Nouvelle protéine ayant une activité de fructosyl-valyl-histidine oxydase et produit modifié de celle-ci et utilisation de la protéine ou du produit modifié
WO2015020200A1 (fr) * 2013-08-09 2015-02-12 キッコーマン株式会社 AMADORIASE MODIFIÉE ET SON PROCÉDÉ DE PRODUCTION, AGENT PERMETTANT D'AMÉLIORER LA RÉSISTANCE AUX TENSIOACTIFS DE L'AMADORIASE ET COMPOSITION PERMETTANT DE MESURER L'HbA1c À L'AIDE DE CELLE-CI
WO2016063984A1 (fr) * 2014-10-24 2016-04-28 キッコーマン株式会社 Amadoriase ayant une activité de déshydrogénase améliorée

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FUJIWARA M. ET AL.: "Alteration of Substrate Specificity of Fructosyl-Amino Acid Oxidase from Ulocladium sp", JS-103, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 102, no. 3, 2006, pages 241 - 243, XP028042246, DOI: 10.1263/jbb.102.241 *

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