Classifications

• • 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.)
• • 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
• • 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 provides fructosyl amino acid oxidase, a method for producing fructosyl amino acid oxidase, a glycated protein sensor comprising fructosyl amino acid oxidase immobilized on a support, and a glycated protein by reaction of fructosyl amino acid oxidase immobilized on a support. It relates to a method for measuring a glycated protein that detects
• Glycated proteins are measured as a management index for diagnosing diabetes and controlling blood sugar. As an example, glycated hemoglobin and glycated albumin are frequently measured in clinical settings.
• Known methods for measuring glycated proteins include electrophoresis, high-efficiency liquid chromatography, immunization, enzymatic methods, and the like.
• a method for measuring a glycated protein by an enzymatic method includes, in the first step, degrading a protein into amino acids or peptides with a protease, and in the second step, glycated amino acids among the amino acids or peptides (hereinafter "glycated amino acids” or “fructosyl amino acids”).
• glycated peptide or a peptide containing glycated amino acids (hereinafter also referred to as “glycated peptide” or “fructosyl peptide”) is allowed to act with fructosyl amino acid oxidase to generate hydrogen peroxide, and third In the step, the hydrogen peroxide is converted into a color-developing reaction and the absorbance is measured, or the electrons released by the hydrogen peroxide being decomposed at the electrode are detected (see Patent Document 1).
• fructosyl amino acid oxidase variants In order to accurately measure specific glycated amino acids and glycated peptides, inventions related to fructosyl amino acid oxidase variants, glycated protein measurement reagents and measurement methods characterized by fructosyl amino acid oxidase are known.
• a fructosyl amino acid oxidase consisting of an amino acid sequence obtained by modifying the amino acid sequence of fructosyl amino acid oxidase of the genus Coniochaeta, acting on fructosyl valyl histidine and substantially acting on other glycated amino acids.
• Hemoglobin A1c is measured by a reaction with fructosyl amino acid oxidase (see Patent Documents 2 and 3) that does not do not and fructosyl amino acid oxidase that satisfies the characteristics that reactivity to fructosyl lysine is 30% or less of reactivity to fructosyl valyl histidine.
• a method (see Patent Document 4), Reagent composition for measuring glycated protein containing fructosyl amino acid oxidase having a property that reactivity to fructosyl lysine is 12 or less when the activity value to fructosyl valyl histidine is 100.
• a first object of the present invention is to provide a novel fructosyl amino acid oxidase.
• a second object of the present invention is to provide a fructosyl amino acid oxidase with excellent physicochemical properties.
• the third object of the present invention is to provide an enzyme that contributes to measurement accuracy and stability in a glycated protein sensor equipped with an immobilized enzyme.
• a fourth object of the present invention is to provide a glycated protein sensor equipped with a novel fructosyl amino acid oxidase and/or a method for measuring glycated protein using the sensor.
• the present inventors discovered a novel fructosyl amino acid oxidase among genes with unknown functions. Furthermore, the present inventors discovered that some of these fructosyl amino acid oxidases have excellent physicochemical properties, and succeeded in imparting these physicochemical properties to another fructosyl amino acid oxidase. bottom.
• the present invention provides wild-type fructosyl amino acid oxidase.
• Another aspect of the present invention provides a fructosyl amino acid oxidase having an amino acid sequence modified so as to have excellent physicochemical properties in measurement of glycated proteins, and a method for producing the same.
• Yet another aspect of the present invention provides a glycated protein sensor equipped with these fructosyl amino acid oxidases and a method for measuring glycated protein.
• the fructosyl amino acid oxidase of the present invention is a fructosyl amino acid oxidase that satisfies one or more of the following (1) to (5).
• (1) contains the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, substituted, added, and/or inserted in the amino acid sequence shown in SEQ ID NO: 1 (2) the above ( (2- 1) When the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 1 are aligned, the amino acid corresponding to position 247 and / or 277 of the amino acid sequence shown in SEQ ID NO: 1 on the amino acid sequence is lysine ( 2-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 430 and/or 443 of the amino acid sequence shown in SEQ ID NO: 1 on the amino acid sequence is lysine.
• the present invention can be a fructosyl amino acid oxidase that satisfies one or more selected from (1) to (3) above and satisfies (4) and/or (5) above.
• (4) and/or (5) may be fructosyl amino acid oxidase immobilized on a support by cross-linking with an amine-reactive cross-linking agent.
• the fructosyl amino acid oxidase of the present invention can be a recombinant protein.
• Another aspect of the present invention is a fructosyl amino acid oxidase that satisfies (2) above and further satisfies one or more selected from (2-3) to (2-4) below.
• (2-3) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 309 and / or 413 of the amino acid sequence shown in SEQ ID NO: 1 on the amino acid sequence is lysine
• the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 424 of the amino acid sequence shown in SEQ ID NO: 1 on the amino acid sequence is lysine.
• Another aspect of the present invention is a fructosyl amino acid oxidase that satisfies (2) above and further satisfies one or more selected from (2-5) to (2-6) below.
• (2-5) Amino acids corresponding to positions 424, 430, and 443 of the amino acid sequence shown in SEQ ID NO: 1 on the amino acid sequence when the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 1 are aligned is lysine
• (2-6) corresponds to positions 309 and 413 of the amino acid sequence shown in SEQ ID NO: 1 on the amino acid sequence when the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 1 are aligned the amino acid is lysine
• the present invention may further be a fructosyl amino acid oxidase that satisfies (4) above.
• (4) above may be a fructosyl amino acid oxidase in a state of being immobilized on a support by cross-linking with an amine-reactive cross-linking agent.
• the amino acid sequence of the fructosyl amino acid oxidase of the present invention exhibits high homology with the amino acid sequence shown in SEQ ID NO: 1, specifically 74% or more.
• Another present invention satisfies all of the above (2-3) to the above (2-4), and furthermore, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the sequence It is a fructosyl amino acid oxidase in which the amino acids corresponding to positions 34, 177, 233, 234 and 297 of the amino acid sequence shown in number 1 are lysines.
• the amino acid sequence of the fructosyl amino acid oxidase of the present invention exhibits high homology with the amino acid sequence shown in SEQ ID NO: 1, specifically 74% or more.
• Another present invention has 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and when the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 1 are aligned, the amino acid sequence shown in SEQ ID NO: A fructosyl amino acid oxidase comprising an amino acid sequence in which the amino acid corresponding to position 247 and/or 277 in the amino acid sequence shown in 1 is lysine, and further satisfying (6-1) and/or (6-2) below. is.
• Another present invention has 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and when the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 1 are aligned, the amino acid sequence shown in SEQ ID NO: 1 contains an amino acid sequence in which the amino acid corresponding to position 247 and / or 277 of the amino acid sequence shown in 1 is lysine, and further has an amino acid substitution described in (7-1) and / or (7-2) below , fructosyl amino acid oxidase.
• the fructosyl amino acid oxidase of the present invention is immobilized on a support compared with the fructosyl amino acid oxidase containing the amino acid sequence before the amino acid substitution described in (7-1) and (7-2) above.
• the fixation to the support may be fixation to the support by cross-linking with an amine-reactive cross-linking agent.
• the heat treatment may be a 48° C. to 80° C. treatment.
• the positions (7-1) and (7-2) of the fructosyl amino acid oxidase of the present invention are positions 56 to 99, 120 to 132, and 155 to 171 of the amino acid sequence shown in SEQ ID NO: 1. , and positions 353 to 404, positions 100 to 119, positions 133 to 154, positions 241 to 288, and positions 435 to 440 of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 1 1 to 55, 172 to 240, 289 to 352, 405 to 434, and 441 to 446 of.
• the fructosyl amino acid oxidase of the present invention has an amino acid sequence that has 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1. It may be a fructosyl amino acid oxidase containing an amino acid sequence that satisfies one or more of the following (a1) to (a6).
• the amino acid at the position corresponding to position 61 of SEQ ID NO: 1 is arginine (a2) The amino acid at the position corresponding to position 67 of SEQ ID NO: 1 is glutamic acid (a3) Corresponding to position 128 of SEQ ID NO: 1 The amino acid at the position is lysine (a4) The amino acid at the position corresponding to position 157 of SEQ ID NO: 1 is lysine (a5) The amino acid at the position corresponding to position 166 of SEQ ID NO: 1 is lysine (a6) SEQ ID NO: The amino acid at the position corresponding to position 391 of 1 is histidine
• Another fructosyl amino acid oxidase of the present invention has an amino acid sequence that has 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 1. It may be a fructosyl amino acid oxidase comprising an amino acid sequence that satisfies one or more of the following (b1) to (b7) when aligned with: (b1) The amino acid at the position corresponding to position 112 of SEQ ID NO: 1 is lysine (b2) The amino acid at the position corresponding to position 115 of SEQ ID NO: 1 is glutamic acid (b3) Corresponding to position 137 of SEQ ID NO: 1 The amino acid at the position is lysine (b4) The amino acid at the position corresponding to position 247 of SEQ ID NO: 1 is lysine (b5) The amino acid at the position corresponding to position 254 of SEQ ID NO: 1 is lysine (b6) SEQ ID NO: The amino
• Another fructosyl amino acid oxidase of the present invention has an amino acid sequence that has 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 1. It may be a fructosyl amino acid oxidase comprising an amino acid sequence that satisfies one or more of the following (c1) to (c11) when aligned with (c1) the amino acid at the position corresponding to position 34 of SEQ ID NO: 1 is lysine (c2) the amino acid at the position corresponding to position 37 of SEQ ID NO: 1 is threonine (c3) corresponding to position 177 of SEQ ID NO: 1 The amino acid at position 1 is lysine (c4) The amino acid at position corresponding to position 198 of SEQ ID NO: 1 is glutamic acid (c5) The amino acid at position corresponding to position 297 of SEQ ID NO: 1 is lysine (c6) SEQ ID NO: The amino acid at the position
• Another fructosyl amino acid oxidase of the present invention has an amino acid sequence that has 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 1. It may be a fructosyl amino acid oxidase comprising an amino acid sequence that satisfies one or more of the following (a1) to (a6), (b1) to (b7), and (c1) to (c11) when aligned with .
• the amino acid at the position corresponding to position 61 of SEQ ID NO: 1 is arginine (a2) The amino acid at the position corresponding to position 67 of SEQ ID NO: 1 is glutamic acid (a3) Corresponding to position 128 of SEQ ID NO: 1 The amino acid at the position is lysine (a4) The amino acid at the position corresponding to position 157 of SEQ ID NO: 1 is lysine (a5) The amino acid at the position corresponding to position 166 of SEQ ID NO: 1 is lysine (a6) SEQ ID NO: The amino acid at the position corresponding to position 391 of SEQ ID NO: 1 is histidine (b1) The amino acid at the position corresponding to position 112 in SEQ ID NO: 1 is lysine (b2) The amino acid at the position corresponding to position 115 in SEQ ID NO: 1 is glutamic acid (b3) The amino acid at the position corresponding to position 137 of SEQ ID NO: 1 is lysine (b4) The amino acid at the position
• the fructosyl amino acid oxidase of the present invention may have fructosyl lysine-specific reactivity.
• the reactivity may be 20 or less to fructosyl valine or a peptide containing fructosyl valine when the reactivity to fructosyl lysine is 100. Further, the reactivity may be 20 or less to fructosylglycine when the reactivity to fructosyllysine is 100.
• the present invention provides a method for producing fructosyl amino acid oxidase.
• a step of substituting lysine for one or more amino acids selected from positions 309, 413, 424, 430, and 443 of the amino acid sequence shown in SEQ ID NO: 1 on the amino acid sequence A step of substituting lysine for one or more amino acids selected from positions 309, 413, 424, 430, and 443 of the amino acid sequence shown in SEQ ID NO: 1 on the amino acid sequence.
• the thermal stability of the fructosyl amino acid oxidase having the amino acid sequence after the amino acid substitution immobilized on the support is compared with the thermal stability of the fructosyl amino acid oxidase having the amino acid sequence before the amino acid substitution immobilized on the support.
• Another method for producing fructosyl amino acid oxidase is to use the following (8-1) to (8-3) in the amino acid sequence of fructosyl amino acid oxidase having 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1.
• Another method for producing fructosyl amino acid oxidase is to use the following (9-1) and (9-2) in the amino acid sequence of fructosyl amino acid oxidase having 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1.
• a step of performing amino acid substitution wherein the thermal stability of the fructosyl amino acid oxidase having the amino acid sequence after the amino acid substitution in a state immobilized on a support is such that the fructosyl amino acid oxidase having the amino acid sequence before the amino acid substitution is
• the thermal stability may be thermal stability in a state of being fixed to the support by cross-linking with an amine-reactive cross-linking agent.
• the fructosyl amino acid oxidase produced by the production method of the present invention has a residual activity after treatment at 48 ° C. in a state fixed to a support, and is treated under the same conditions in a state not fixed to a support.
• the residual activity may be higher than that after treatment, and/or the residual activity after treatment at 65° C. to 80° C. for 20 minutes while immobilized on a support may be 70% or more.
• the amino acid sequence homology in the production method of the present invention may be 74% or more.
• the present invention also provides a glycated protein sensor comprising a support and fructosyl amino acid oxidase immobilized on the support, wherein the fructosyl amino acid oxidase is immobilized on the support.
• a glycated protein sensor having a residual activity of 70% or more after 20-minute treatment at °C to 80°C.
• a fructosyl amino acid oxidase may be immobilized on a support by cross-linking with an amine-reactive cross-linker.
• Another aspect of the present invention is a glycated protein sensor comprising a support and fructosyl amino acid oxidase immobilized on the support, wherein the fructosyl amino acid oxidase is the fructosyl amino acid oxidase described above.
• a glycated protein sensor is provided.
• a fructosyl amino acid oxidase may be immobilized on a support by cross-linking with an amine-reactive cross-linker.
• the present invention provides a glycated protein sensor further comprising a hydrogen peroxide detection unit.
• a support layer containing a support and a hydrogen peroxide detection part may be laminated.
• the present invention provides a method for measuring glycated proteins that detects glycated proteins through the reaction of fructosyl amino acid oxidase immobilized on a support.
• the fructosyl amino acid oxidase of the present invention has specific reactivity to specific glycated amino acids and/or specific glycated peptides.
• the fructosyl amino acid oxidase of the present invention the amount of a specific glycated amino acid and/or specific glycated peptide contained in a test sample can be measured accurately.
• thermostability of the fructosyl amino acid oxidase immobilized on the support can be improved, so the long-term stability of the glycated protein sensor containing the enzyme is improved.
• the glycated protein sensor of the present invention is advantageous in that highly reliable measurement results can be obtained over a long period of time because the decrease in enzymatic activity during storage is suppressed.
• FIG. 2 shows the results of multiple alignment analysis of the amino acid sequences of fructosyl amino acid oxidases of Examples and Comparative Examples.
• FIG. 2 shows the results of multiple alignment analysis of the amino acid sequences of fructosyl amino acid oxidases of Examples and Comparative Examples.
• 1 is a schematic diagram of a glycated protein sensor 10 that is one embodiment of the present invention.
• FIG. FIG. 2 is a schematic diagram of a glycated protein sensor 20, another embodiment of the present invention.
• 1 is a drawing showing the results of SDS-PAGE of fructosyl amino acid oxidase in Examples. It is a drawing showing the results of evaluating the thermal stability in liquid phase of the fructosyl amino acid oxidases of Examples.
• 1 is a drawing showing the results of evaluating the thermal stability of fructosyl amino acid oxidases of Examples in a state of being immobilized on a support.
• 1 is a drawing showing the results of evaluating the thermal stability of fructosyl amino acid oxidases of Examples in a state of being immobilized on a support.
• 1 is a drawing showing the results of evaluating the thermal stability of fructosyl amino acid oxidases and fructosyl amino acid oxidase variants of Examples in a state of being immobilized on a support.
• the fructosyl amino acid oxidase of the present invention is a novel fructosyl amino acid oxidase.
• the novel fructosyl amino acid oxidases of the present invention include highly thermostable fructosyl amino acid oxidases.
• the present invention provides a method for producing a fructosyl amino acid oxidase modified to enhance thermostability.
• the present invention provides a glycated protein sensor comprising a support and fructosyl amino acid oxidase immobilized on the support, and a glycated protein sensor for detecting glycated proteins by reaction of the fructosyl amino acid oxidase immobilized on the support.
• a method for measuring protein is provided.
• BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below based on embodiments.
• Fructosyl amino acid oxidase is an enzyme that acts on an amino acid having a glycated ⁇ -amino group and/or an ⁇ -amino group, or a peptide containing the amino acid, and produces hydrogen peroxide in the process of deglycosylating the glycated amino acid.
• Fructosylamino acid oxidase (hereinafter sometimes referred to as "FAOD”) is also called amadoriase, ketoamine oxidase, and fructosylamine oxidase.
• Fructosyl amino acid oxidase includes fructosyl peptide oxidase (hereinafter also referred to as "FPOD" or "FPOX”) that acts on glycated peptides.
• Fructosyl amino acid oxidase uses glycated amino acids and/or glycated peptides as substrates.
• Specific examples of glycated amino acids include ⁇ -fructosyllysine (sometimes referred to as “fructosyllysine”) and ⁇ -fructosylvaline (sometimes referred to as “fructosylvaline”).
• ⁇ -fructosylglycine (sometimes referred to as “fructosylglycine”)
• ⁇ -fructosylhistiditin (sometimes referred to as “fructosylhistidine”)
• ⁇ -fructosylleucine (sometimes referred to as "fructosylleucine")
• ⁇ -fructosylserine (sometimes referred to as "fructosylserine”), and the like.
• the glycated peptide is a peptide consisting of 2 to 10 amino acids, preferably 2 to 6 amino acids, more preferably 2 to 3 amino acids, wherein one or more glycated amino acids and a peptide comprising Examples thereof include ⁇ -fructosyl-valyl histidine (sometimes referred to as "fructosyl-valyl histidine").
• the fructosyl amino acid oxidase of the present invention has the following physicochemical properties. It has an optimum pH range of pH 7-9, an action pH range of pH 5-9, an action temperature of 20-80° C., and is soluble in buffer solutions. Specific examples of buffers include Tris-hydrochloride buffer, phosphate-buffered saline (PBS), and the like. Also, the molecular weight of the fructosyl amino acid oxidase of the present invention on SDS-PAGE can be about 45 kDa to about 55 kDa, preferably about 48 kDa to about 50 kDa.
• the fructosyl amino acid oxidase of one embodiment of the present invention is stable against heat while immobilized on a support.
• a fructosyl amino acid oxidase that is stable against heat while being immobilized on a support means a fructosyl amino acid oxidase that has a high residual activity after being heat-treated while being immobilized on a support, and has a temperature of 65° C. or higher.
• One example is fructosyl amino acid oxidase, which is characterized by a residual activity of 70% or more after treatment.
• the treatment temperature can be 48°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C.
• the treatment time is 10 to 30 minutes, preferably 15 to 25 minutes, most preferably 20 minutes.
• fructosyl amino acid oxidase that is stable against heat when immobilized on a support
• the residual activity after heat treatment when immobilized on a support is fructosyl amino acid oxidase with higher residual activity after treatment under the same conditions.
• the treatment temperature is 48°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C.
• the treatment time is 10 to 30 minutes, preferably 15 to 25 minutes, most preferably 20 minutes.
• the fructosyl amino acid oxidase-immobilized support is heated by known means such as an electric oven or a constant temperature bath, and the residual activity of fructosyl amino acid oxidase is measured.
• the residual activity refers to the ratio of the enzyme activity after heat treatment at each temperature to the enzyme activity before heat treatment.
• Examples of means for immobilizing enzymes such as fructosyl amino acid oxidase include covalent bond methods, physical adsorption methods, ionic bond methods, cross-linking methods, entrapment methods, and biochemical specific binding methods. Depending on the enzyme used, an immobilization method that does not deactivate the enzyme may be selected, or multiple immobilization methods may be used in combination.
• the enzyme is immobilized on the support by cross-linking with a cross-linking agent, alone or in a carrier-bound state.
• the support is a protein different from the enzyme, and the enzyme is immobilized on the support using a cross-linking agent such as glutaraldehyde or an isocyanate derivative after mixing the enzyme and the support.
• proteins include albumins such as bovine serum albumin (BSA), collagen, and gelatin.
• the support may be the same enzyme as the immobilized enzyme. In such cases, also called self-aggregation, enzymes are immobilized on the same enzyme by cross-linking with a cross-linking agent.
• Cross-linking agents used are substances that cross-link intermolecularly or intramolecularly, and specific examples include glutaraldehyde, isocyanate derivatives, formaldehyde, glyoxal, malondialdehyde, and succinaldehyde. Among them, an amine-reactive cross-linking agent is preferably used as the cross-linking agent.
• Amine-reactive cross-linking agents are cross-linking agents that react with amino groups of proteins to cross-link proteins, and specific examples include glutaraldehyde, formaldehyde, N-hydroxyesters, amide esters, imidoesters, and the like.
• the amine-reactive cross-linking agent reacts with amino groups present on the surface of fructosyl amino acid oxidase and fixes the fructosyl amino acid oxidase and the support by cross-linking.
• the fructosyl amino acid oxidase can be heat stable while immobilized on a support.
• synthetic polymers resins, inorganic materials, polysaccharides, minerals, clay, etc. may be used for the support.
• synthetic polymers resins, inorganic materials, polysaccharides, minerals, clay, etc.
• a fluororesin, an ion exchange resin, a polyvinyl alcohol resin, a hydraulic resin, a photocurable resin, a solid polymer electrolyte, a polyion complex, urethane, or the like is used as a support, chemical properties such as hydrophobicity may occur.
• Supports can also be beads.
• the beads may be carbon microparticles (carbon beads), silica (SiO 2 ) microparticles (silica beads), or beads made of polysaccharides such as chitin, chitosan, and alginic acid.
• the beads may contain fine metal particles or a magnetizable substance, and may be magnetic beads.
• the beads may have an average particle size of 10 nm or more and may be 200 nm or less. Enzymes can be immobilized by being cross-linked to the beads.
• the fructosyl amino acid oxidase of the present invention can react specifically with a specific glycated amino acid or a peptide containing that amino acid.
• the fructosyl amino acid oxidase is highly specific for fructosyl lysine.
• High specificity to fructosyl lysine means reaction to other glycated amino acids or peptides containing other glycated amino acids when reactivity to fructosyl lysine or peptides containing fructosyl lysine is 100
• the reactivity is said to be 20 or less
• the reactivity to other glycated amino acids or peptides comprising other glycated amino acids here is preferably 10 or less, more preferably 5 or less, It is more preferably 1 or less.
• glycated amino acids refer to one or more selected from, for example, fructosylvaline, fructosylglycine, fructosylhistidine, fructosylleucine, and fructosylserine.
• Fructosyl amino acid oxidases are classified into three groups based on substrate specificity. Fructosyl amino acid oxidases belonging to Group 1 have high specificity for amino acids whose ⁇ -amino groups are glycated and/or peptides containing such amino acids. Fructosyl amino acid oxidases belonging to Group 2 have high specificity for amino acids with glycosylated ⁇ -amino groups and/or peptides containing such amino acids.
• a fructosyl amino acid oxidase belonging to Group 3 has specificity for an amino acid having a glycated ⁇ -amino group and/or a peptide containing the amino acid, and an amino acid having a glycated ⁇ -amino group and/or a peptide containing the amino acid. expensive.
• the fructosyl amino acid oxidase of the present invention can be a fructosyl amino acid oxidase belonging to any group.
• fructosyl amino acid oxidase has high specificity for fructosyl valyl histidine.
• High specificity to fructosyl valyl histidine means that the reactivity to other glycated amino acids or other glycated peptides is 20 or less when the reactivity to fructosyl valyl histidine is 100.
• the reactivity to other glycated amino acids or other fructoside peptides is preferably 10 or less, more preferably 5 or less, and even more preferably 1 or less.
• glycated amino acids refer to one or more selected from, for example, fructosyl valine, fructosyl lysine, fructosyl glycine, fructosyl histidine, fructosyl leucine, and fructosyl serine, and other fructosyl peptides , refers to peptides comprising glycated amino acids, other than fructosyl valyl histidine.
• the fructosyl amino acid oxidase of the present invention can react specifically with a specific glycated amino acid or a peptide containing that amino acid.
• the fructosyl amino acid oxidase is highly specific for fructosyl valine.
• High specificity to fructosyl valine means that when the reactivity to fructosyl valine or a peptide containing fructosyl valine is 100, it contains other glycated amino acids or other glycated amino acids
• the reactivity to peptides is said to be 20 or less, but the reactivity to other glycated amino acids or peptides containing other glycated amino acids is preferably 10 or less, and preferably 5 or less. It is more preferably 1 or less.
• glycated amino acids refer to one or more selected from, for example, fructosyllysine, fructosylglycine, fructosylhistidine, fructosylleucine, and fructosylserine.
• the fructosyl amino acid oxidase of the present invention can be a recombinant protein expressed by introducing a nucleic acid encoding the fructosyl amino acid oxidase of the present invention into a host such as E. coli, fungi, yeast, mammalian cells, insect cells, and the like.
• a host such as E. coli, fungi, yeast, mammalian cells, insect cells, and the like.
• fructosyl amino acid oxidase can be used not only by cloning from microorganisms, animals, plants, etc., but also by artificially mutating, modifying, or designing.
• the inventors discovered a novel fructosyl amino acid oxidase among genes with unknown functions. Among them, a fructosyl amino acid oxidase having remarkably high thermal stability in a state immobilized on a support was found. and succeeded in increasing the thermal stability.
• Table 1 shows the amino acid sequences of the novel fructosyl amino acid oxidases found by the inventors.
• the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1 is derived from Aspergillus pseudotamarii of the genus Aspergillus. Until now, it was expected to be one of the FAD-dependent oxidoreductases [Accession Number: XP_031920077] from the sequence homology, but it was not known that the protein could function as a fructosyl amino acid oxidase.
• the present inventors have found that the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1 has remarkably high thermal stability when immobilized on a support, and has specific glycated amino acids and/or glycated peptides. It was found that the substrate specificity for
• the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 2 is derived from Penicillium rubens of the genus Penicillium. Until now, it was known as a hypothetical protein [Accession Number: XP_002568014], but it was not known that the protein could function as a fructosyl amino acid oxidase. The present inventors have found that the fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 2 has extremely high substrate specificity for specific glycated amino acids and/or glycated peptides.
• the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 3 is derived from Penicillium flavigenum of the genus Penicillium. Until now, it was known as a hypothetical protein [Accession Number: OQE32666], but it was not known that the protein could function as a fructosyl amino acid oxidase. The present inventors have found that the fructosylamino acid oxidase having the amino acid sequence shown in SEQ ID NO: 3 has extremely high substrate specificity for specific glycated amino acids and/or glycated peptides.
• the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 4 is derived from Monascus purpureus of the genus Monascus. Until now, it was known as a hypothetical protein [Accession Number: TQB73262], but it was not known that the protein could function as a fructosyl amino acid oxidase. The present inventors have found that the fructosylamino acid oxidase having the amino acid sequence shown in SEQ ID NO: 4 has extremely high substrate specificity for specific glycated amino acids and/or glycated peptides.
• the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 5 is derived from Coniochaeta pulveracea of the genus Coniochaeta. Until now, it was known as a hypothetical protein [Accession Number: RKU49498], but it was not known that the protein could function as a fructosyl amino acid oxidase. The present inventors have found that the fructosylamino acid oxidase having the amino acid sequence shown in SEQ ID NO: 5 has extremely high substrate specificity for specific glycated amino acids and/or glycated peptides.
• the present invention also provides a fructosyl amino acid oxidase having an amino acid sequence highly homologous to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, which has high thermal stability when immobilized on a support, and specific
• a fructosyl amino acid oxidase having physicochemical properties such as high substrate specificity for glycated amino acids and glycated peptides can also be used.
• the amino acid sequence of fructosyl amino acid oxidase is 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 51% or more, 52% or more of the amino acid sequence of SEQ ID NO: 1, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, It may contain amino acid sequences with 78% or more, 79% or more, or 80% or more homology. These homology numbers are rounded to one decimal place.
• the fructosyl amino acid oxidase provided in the glycated protein sensor can be SEQ ID NO: 1 or a fructosyl amino acid oxidase consisting of an amino acid sequence other than SEQ ID NO: 1.
• a fructosyl amino acid oxidase consisting of an amino acid sequence other than SEQ ID NO: 1 is a variant of fructosyl amino acid oxidase, and has high thermal stability when immobilized on a support, and high heat stability against specific glycated amino acids and glycated peptides. Physicochemical properties such as substrate specificity may be imparted.
• the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1 has extremely high stability against heat when immobilized on a support, and has a temperature range of 65° C. to 80° C. when immobilized on a support.
• the residual activity after treatment for 20 minutes at ° C. is 70% or more, and / or the residual activity after heat treatment in the state fixed to the support is the same condition without being fixed to the support. It was found to exhibit excellent physicochemical properties such as higher residual activity after treatment.
• fructosyl consisting of an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NO: 1 and satisfying the following requirements (1) and (2) It is an amino acid oxidase and can also be a fructosyl amino acid oxidase containing the amino acid sequence.
• (1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 247 and/or 277 of the amino acid sequence shown in SEQ ID NO: 1 on the amino acid sequence is lysine.
• SEQ ID NOS: 24 and 25 are known amino acid sequences of fructosyl amino acid oxidase.
• SEQ ID NO: 24 is Aspergillus oryzae-derived fructosyl amino acid oxidase [Accession Number: BAD54824]
• SEQ ID NO: 25 is Aspergillus fumigatus-derived fructosyl amino acid oxidase [Accession Number: AAB88209].
• amino acids at positions 247, 277, 430 and 443 of the amino acid sequence shown in SEQ ID NO: 1 are indicated by arrows.
• Table 2 shows the amino acids in SEQ ID NOS:2-5, SEQ ID NO:24, and SEQ ID NO:25 that correspond to lysines at positions 247, 277, 430, and 443 of the amino acid sequence shown in SEQ ID NO:1.
• the amino acid sequences satisfying (1) and (2) above are distinguished from SEQ ID NOS: 24 and 25, which are known fructosyl amino acid oxidase amino acid sequences.
• lysines at positions 309, 413, and 424 in the amino acid sequence of SEQ ID NO: 1 also play an important role in thermostability of the enzyme. That is, in one embodiment of the present invention, an amino acid sequence having a high homology with the amino acid sequence shown in SEQ ID NO: 1, when the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 1 are aligned, the amino acid sequence A fructosyl amino acid oxidase consisting of an amino acid sequence in which the amino acid corresponding to positions 309, 413 and/or 424 of the amino acid sequence shown in SEQ ID NO: 1 is lysine, and a fructosyl amino acid comprising the amino acid sequence. It can also be an oxidase.
• the amino acids at positions 309, 413 and 424 of the amino acid sequence shown in SEQ ID NO: 1 are indicated by arrowheads.
• lysines at positions 34, 177, 233, 234, and 297 in the amino acid sequence of SEQ ID NO: 1 also play an important role in thermostability of the enzyme. It became clear. That is, in one embodiment of the present invention, an amino acid sequence having a high homology with the amino acid sequence shown in SEQ ID NO: 1, when the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 1 are aligned, the amino acid sequence A fructosyl amino acid oxidase consisting of an amino acid sequence in which the amino acid corresponding to positions 34, 177, 233, 234 and/or 297 of the amino acid sequence shown in SEQ ID NO: 1 is lysine, and the amino acid thereof It can also be a fructosyl amino acid oxidase containing sequence.
• amino acids at positions 34, 177, 233, 234 and 297 of the amino acid sequence shown in SEQ ID NO: 1 are shown with white arrowheads
• a fructosyl amino acid oxidase comprising an amino acid sequence and may be a fructosyl amino acid oxidase comprising the amino acid sequence.
• the amino acid corresponding to one or more selected from positions 166, 167, 394 and 397 is lysine.
• the amino acids corresponding to positions 166, 167, 394 and 397 are lysines.
• the present invention also provides a method for producing a fructosyl amino acid oxidase containing a modified amino acid sequence.
• the method for producing fructosyl amino acid oxidase of the present invention includes a step of substituting an amino acid in the amino acid sequence of fructosyl amino acid oxidase.
• amino acid sequence before modification an amino acid sequence of fructosyl amino acid oxidase having high homology with the amino acid sequence shown in SEQ ID NO: 1 (hereinafter referred to as "amino acid sequence before modification"), SEQ ID NO: 1
• amino acid sequence before modification amino acid sequence before modification
• positions 34, 64, 84, 87, 112, 177, 128, 137, 152, 157, 166, 167 of SEQ ID NO: 1 Equivalent to 233rd, 234th, 247th, 254th, 255th, 267th, 277th, 297th, 309th, 394th, 397th, 413th, 424th, 430th, 438th and 443rd It may be a step of substituting lysine for one or more amino acids on the amino acid sequence before modification.
• the fructosyl amino acid oxidase modified by the production method of the present invention has higher thermostability than the fructosyl amino acid oxidase before modification. More preferably, the fructosyl amino acid oxidase modified by the production method of the present invention has higher thermostability when immobilized on a support than the fructosyl amino acid oxidase before modification.
• the present inventors divided SEQ ID NO: 1 into three regions [(a), (b) and (c)] based on the three-dimensional structure of fructosyl amino acid oxidase, and modified amino acids in each region. , can improve the physicochemical properties of fructosyl amino acid oxidase.
• region (a) is a region consisting of amino acids corresponding to positions 56 to 99, 120 to 132, 155 to 171, and 353 to 404 of SEQ ID NO: 1
• region (b) is , a region consisting of amino acids corresponding to positions 100 to 119, 133 to 154, 241 to 288, and 435 to 440 of SEQ ID NO: 1
• region (c) is 1 to 1 of SEQ ID NO: 1 It is a region consisting of amino acids corresponding to positions 55, 172-240, 289-352, 405-434, and 441-446.
• Another embodiment of the method for producing a fructosyl amino acid oxidase of the present invention is a fructosyl amino acid oxidase comprising an amino acid sequence highly homologous to SEQ ID NO: 1, for each region, the following (1) and (2) Including modifying the amino acid to meet the requirements.
• an enzyme having higher thermostability than the enzyme before modification can be obtained.
• alignment of amino acid sequences can be performed using programs such as Blast, Clustal W, and Clustal Omega. Alignment provides information such as homology, identity or similarity between a plurality of amino acid sequences, positions of corresponding amino acids, and the like.
• the fructosyl amino acid oxidase has the following (a1) to (a6), (b1) to (b7), and (c1) to ( A fructosyl amino acid oxidase modified to satisfy one or more selected from the group consisting of c11), wherein the fructosyl amino acid oxidase has higher thermal stability when immobilized on a support than the fructosyl amino acid oxidase before modification. It is an improved fructosyl amino acid oxidase.
• the amino acid at the position corresponding to position 61 of SEQ ID NO: 1 is arginine (a2) The amino acid at the position corresponding to position 67 of SEQ ID NO: 1 is glutamic acid (a3) Corresponding to position 128 of SEQ ID NO: 1 The amino acid at the position is lysine (a4) The amino acid at the position corresponding to position 157 of SEQ ID NO: 1 is lysine (a5) The amino acid at the position corresponding to position 166 of SEQ ID NO: 1 is lysine (a6) SEQ ID NO: The amino acid at the position corresponding to position 391 of SEQ ID NO: 1 is histidine (b1) The amino acid at the position corresponding to position 112 in SEQ ID NO: 1 is lysine (b2) The amino acid at the position corresponding to position 115 in SEQ ID NO: 1 is glutamic acid (b3) The amino acid at the position corresponding to position 137 of SEQ ID NO: 1 is lysine (b4) The amino acid at the position
• amino acid sequences of modified fructosyl amino acid oxidases are shown.
• the amino acid sequence shown in SEQ ID NO:6 is a modification of the amino acid sequence shown in SEQ ID NO:2.
• the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 6.
• (a1) Lysine at position 61 of SEQ ID NO: 2 (corresponding to position 61 of SEQ ID NO: 1) was replaced with arginine.
• (a2) Lysine at position 67 of SEQ ID NO: 2 (corresponding to position 67 of SEQ ID NO: 1) was substituted with glutamic acid.
• the amino acid sequence shown in SEQ ID NO:7 is a modification of the amino acid sequence shown in SEQ ID NO:3.
• the amino acid sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 7.
• (a1) Lysine at position 61 of SEQ ID NO: 3 (corresponding to position 61 of SEQ ID NO: 1) was replaced with arginine.
• (a2) Lysine at position 67 of SEQ ID NO: 3 (corresponding to position 67 of SEQ ID NO: 1) was substituted with glutamic acid.
• the amino acid sequence shown in SEQ ID NO:8 is a modification of the amino acid sequence shown in SEQ ID NO:4.
• the amino acid sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 8.
• (a1) Lysine at position 60 of SEQ ID NO: 4 (corresponding to position 61 of SEQ ID NO: 1) was replaced with arginine.
• Arginine at position 159 of SEQ ID NO: 4 (corresponding to position 166 of SEQ ID NO: 1) was replaced with lysine.
• the amino acid sequence shown in SEQ ID NO:9 is a modification of the amino acid sequence shown in SEQ ID NO:5.
• the amino acid sequence shown in SEQ ID NO: 5 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 9.
• Arginine at position 163 of SEQ ID NO: 5 (corresponding to position 166 of SEQ ID NO: 1) was replaced with lysine.
• Lysine at position 387 of SEQ ID NO: 5 was substituted with histidine.
• the amino acid sequence shown in SEQ ID NO: 10 is a modified version of the amino acid sequence shown in SEQ ID NO: 2.
• the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 10.
• (b1) Glutamic acid at position 112 of SEQ ID NO: 2 (corresponding to position 112 of SEQ ID NO: 1) was substituted with lysine.
• (b2) Lysine at position 115 of SEQ ID NO: 2 (corresponding to position 115 of SEQ ID NO: 1) was substituted with glutamic acid.
• the amino acid sequence shown in SEQ ID NO: 11 is a modified version of the amino acid sequence shown in SEQ ID NO: 3.
• the amino acid sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 11.
• (b1) Glutamic acid at position 112 of SEQ ID NO: 3 (corresponding to position 112 of SEQ ID NO: 1) was substituted with lysine.
• (b2) Lysine at position 115 of SEQ ID NO: 3 (corresponding to position 115 of SEQ ID NO: 1) was substituted with glutamic acid.
• the amino acid sequence shown in SEQ ID NO: 12 is a modified version of the amino acid sequence shown in SEQ ID NO: 4.
• the amino acid sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 12.
• (b1) Lysine was substituted for glutamic acid at position 105 of SEQ ID NO: 4 (corresponding to position 112 of SEQ ID NO: 1).
• the amino acid sequence shown in SEQ ID NO: 13 is a modified version of the amino acid sequence shown in SEQ ID NO: 5.
• the amino acid sequence shown in SEQ ID NO: 5 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 13.
• (b1) Glutamic acid at position 109 of SEQ ID NO: 5 (corresponding to position 112 of SEQ ID NO: 1) was substituted with lysine.
• Lysine was substituted for glycine at position 134 of SEQ ID NO: 5 (corresponding to position 137 of SEQ ID NO: 1).
• the amino acid sequence shown in SEQ ID NO: 14 is a modified version of the amino acid sequence shown in SEQ ID NO: 2.
• the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 14.
• (c1) Arginine at position 34 of SEQ ID NO: 2 (corresponding to position 34 of SEQ ID NO: 1) was replaced with lysine.
• (c2) Lysine at position 37 of SEQ ID NO: 2 (corresponding to position 37 of SEQ ID NO: 1) was replaced with threonine.
• Lysine was substituted for aspartic acid at position 430 of SEQ ID NO: 2 (corresponding to position 430 of SEQ ID NO: 1).
• Lysine at position 432 of SEQ ID NO: 2 was replaced with glycine.
• Methionine at position 443 of SEQ ID NO: 2 was replaced with lysine.
• the amino acid sequence shown in SEQ ID NO: 15 is a modified version of the amino acid sequence shown in SEQ ID NO: 3.
• the amino acid sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 15.
• (c2) Lysine at position 37 of SEQ ID NO: 3 (corresponding to position 37 of SEQ ID NO: 1) was replaced with threonine.
• Glutamine at position 177 of SEQ ID NO: 3 (corresponding to position 177 of SEQ ID NO: 1) was replaced with lysine.
• the amino acid sequence shown in SEQ ID NO: 16 is a modified version of the amino acid sequence shown in SEQ ID NO: 4.
• the amino acid sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 16.
• Lysine was substituted for asparagine at position 292 of SEQ ID NO: 4 (corresponding to position 297 of SEQ ID NO: 1).
• (c6) Lysine at position 297 of SEQ ID NO: 4 (corresponding to position 302 of SEQ ID NO: 1) was replaced with valine.
• Lysine was substituted for glutamic acid at position 425 of SEQ ID NO: 4 (corresponding to position 430 of SEQ ID NO: 1).
• Serine at position 438 of SEQ ID NO: 4 was replaced with lysine.
• the amino acid sequence shown in SEQ ID NO: 17 is a modified version of the amino acid sequence shown in SEQ ID NO: 5.
• the amino acid sequence shown in SEQ ID NO: 5 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 17.
• Lysine was substituted for glycine at position 294 of SEQ ID NO: 5 (corresponding to position 297 of SEQ ID NO: 1).
• Lysine was substituted for glutamic acid at position 426 of SEQ ID NO: 5 (corresponding to position 430 of SEQ ID NO: 1).
• the amino acid sequence shown in SEQ ID NO: 18 is a modified version of the amino acid sequence shown in SEQ ID NO: 2.
• the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 18.
• (a1) Lysine at position 61 of SEQ ID NO: 2 (corresponding to position 61 of SEQ ID NO: 1) was replaced with arginine.
• (a2) Lysine at position 67 of SEQ ID NO: 2 (corresponding to position 67 of SEQ ID NO: 1) was substituted with glutamic acid.
• the amino acid sequence shown in SEQ ID NO: 19 is a modified version of the amino acid sequence shown in SEQ ID NO: 3.
• the amino acid sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 19.
• (a1) Lysine at position 61 of SEQ ID NO: 3 (corresponding to position 61 of SEQ ID NO: 1) was replaced with arginine.
• (a2) Lysine at position 67 of SEQ ID NO: 3 (corresponding to position 67 of SEQ ID NO: 1) was substituted with glutamic acid.
• the amino acid sequence shown in SEQ ID NO:20 is a modified version of the amino acid sequence shown in SEQ ID NO:4.
• the amino acid sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 20.
• (a1) Lysine at position 60 of SEQ ID NO: 4 (corresponding to position 61 of SEQ ID NO: 1) was replaced with arginine.
• the amino acid sequence shown in SEQ ID NO:21 is a modified version of the amino acid sequence shown in SEQ ID NO:5.
• the amino acid sequence shown in SEQ ID NO: 5 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 21.
• Lysine was substituted for proline at position 154 of SEQ ID NO: 5 (corresponding to position 157 of SEQ ID NO: 1).
• Arginine at position 163 of SEQ ID NO: 5 (corresponding to position 166 of SEQ ID NO: 1) was replaced with lysine.
• Tables 3 to 6 show the sequence numbers of the amino acid sequences of the fructosyl amino acid oxidase before modification and the fructosyl amino acid oxidase after modification, and the amino acids substituted in the amino acid sequences after modification and their positions. For example, substitution of arginine (R) at position 34 with lysine (K) is represented as "R34K.”
• the amino acid sequence shown in SEQ ID NO: 22 is obtained by modifying the amino acid sequence (SEQ ID NO: 24) of known FAOD [Accession Number: BAD54824] derived from Aspergillus oryzae.
• the amino acid sequence shown in SEQ ID NO:24 and the amino acid sequence shown in SEQ ID NO:1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO:22.
• Lysine was substituted for asparagine at position 277 of SEQ ID NO: 24 (corresponding to position 277 of SEQ ID NO: 1) (N277K).
• the amino acid sequence shown in SEQ ID NO: 23 is a modified amino acid sequence (SEQ ID NO: 25) of known FAOD [Accession Number: AAB88209] derived from Aspergillus fumigatus.
• the amino acid sequence shown in SEQ ID NO: 25 and the amino acid sequence shown in SEQ ID NO: 1 were aligned and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO: 23.
• the fructosyl amino acid oxidase whose amino acids have been modified as described above has improved thermal stability when immobilized on a support compared to fructosyl amino acid oxidase before modification.
• a recombinant protein of fructosyl amino acid oxidase consisting of these amino acid sequences can be used.
• the fructosyl amino acid oxidase that can be used in the present invention includes Escherichia coli, Saccharomyces cerevisiae, and methylotrophic yeast ( Pichia pastoris), mammalian cells, insect cells, etc., are used as hosts, and the hosts are cultured, separated from fungal bodies and cells, and subjected to protein purification, concentration and other treatments.
• Another embodiment of the present invention is an amino acid sequence in which one or several amino acids are modified or mutated, or deleted, substituted, added and / or inserted in the amino acid sequence shown in any of SEQ ID NOS: 1 to 23. It is a fructosyl amino acid oxidase that contains glycosylated amino acids and has physicochemical properties such as high thermal stability when immobilized on a support and high substrate specificity for specific glycated amino acids and glycated peptides.
• one or several amino acids means 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 amino acids.
• the present invention further provides a glycated protein sensor.
• the glycated protein sensor of the present invention comprises at least a support and an enzyme immobilized on the support. Enzymes include at least fructosyl amino acid oxidase.
• the glycated protein sensor of the present invention can be a glycated protein sensor that calculates the amount of glycated protein contained in a test sample, that is, the concentration of glycated protein in the test sample.
• a preferred embodiment of the glycated protein sensor of the present invention comprises a support, fructosyl amino acid oxidase immobilized on the support, and a detection part.
• the detection unit detects hydrogen peroxide produced from glycated amino acids and/or glycated peptides by fructosyl amino acid oxidase.
• the glycated protein sensor can calculate the amount (concentration) of the glycated protein from the detection result of the detection unit.
• the detection unit is a hydrogen peroxide detection unit that detects hydrogen peroxide
• the detection unit can be a hydrogen peroxide electrode. Since the hydrogen peroxide electrode detects the electrons emitted when hydrogen peroxide is decomposed into oxygen as a current, the amount (concentration) of hydrogen peroxide can be calculated from the detected current value. .
• the detection unit is an optical detection unit that detects hydrogen peroxide by measuring absorbance and light intensity.
• hydrogen peroxide is quantified by detecting a color reaction in the presence of peroxidase and an oxidative coloring dye, or by detecting the luminescence intensity of luminol.
• the detection part is an electrochemiluminescence detection part, and a gold electrode, a platinum electrode, or a transparent electrode of indium tin oxide (ITO electrode) is used.
• Hydrogen peroxide is detected by measuring luminescence with a luminescent reagent such as luminol.
• the detector may be another type of hydrogen peroxide detector.
• the test sample of the glycated protein sensor of the present invention can be a solution.
• the solution may be a body fluid, a solution derived from the body fluid, or a diluted solution of the body fluid.
• the solution may be a solution that is not a bodily fluid (derived from a non-bodily fluid), or a mixture of a bodily fluid or a bodily fluid-derived solution and a non-bodily fluid-derived solution.
• the solution may be the solution used for sample measurements or the solution used for calibration measurements.
• the solution may be a standard solution or a calibrator solution.
• the solution may contain a buffer.
• the body fluid may be blood, serum, plasma, lymph, tissue fluid such as interstitial fluid, interstitial fluid, interstitial fluid, body cavity fluid, serous fluid, pleural fluid, ascites, pericardial fluid, It may be cerebrospinal fluid (cerebrospinal fluid), synovial fluid (synovial fluid), or aqueous humor (aqueous humor).
• the bodily fluid may be a digestive fluid such as saliva, gastric juice, bile, pancreatic juice, intestinal juice, sweat, tears, runny nose, urine, semen, vaginal fluid, amniotic fluid, milk.
• the bodily fluid may be an animal bodily fluid or a human bodily fluid.
• a bodily fluid may be a liquid in a food containing protein of animal origin (eg, milk or dairy products).
• the body fluid may be a plant body fluid, a plant biological fluid, or a plant-derived fluid.
• the bodily fluid may be the juice, nectar, or sap of a plant.
• the solution may contain the substance to be measured.
• the solution may be tears, and the substance to be measured may be albumin or glycoalbumin contained in tears.
• the object to be measured may be albumin, glycoalbumin, hemoglobin, glycohemoglobin in blood, serum or plasma, albumin in interstitial fluid, glycoalbumin, albumin in urine, glycosylation It may be albumin, albumin in saliva, or glycoalbumin.
• the measurement target of the glycated protein sensor of the present invention is glycated protein.
• the glycated protein may be fructosamine.
• Fructosamine is a general term for glycated proteins contained in blood, and specifically includes glycated albumin and glycated hemoglobin contained in blood.
• a support-immobilized protease may also be included.
• Protease is a general term for peptide bond hydrolases that hydrolyze and catabolize proteins and polypeptides.
• a protease may be an enzyme that breaks proteins into peptide fragments.
• the protein contains glycated amino acid residues, one or more selected from the group consisting of glycated amino acids, peptide fragments containing glycated amino acids, non-glycated amino acids, and peptide fragments not containing glycated amino acids by the action of proteases can occur.
• Fructosyl amino acid oxidase can react with glycated amino acids, or peptide fragments containing glycated amino acids, among others, to produce hydrogen peroxide.
• the enzyme-immobilized support forms a support layer such as a thin film layer, and is stacked on the detection surface of the detection unit.
• a bonding agent such as a silane coupling agent is used to position the support on or near the detection surface of the detection unit.
• a bonding layer is formed between the layered support and the detection section, thereby bonding the support and the detection surface.
• Bonding agents may include, for example, materials that bond inorganic and organic materials.
• the bonding agent may be, for example, a silane coupling agent. Examples of silane coupling agents include the following.
• Vinyl type vinyltrimethoxysilane, vinyltriethoxysilane, 7-octenyltrimethoxysilane, vinyldimethylethoxysilane, vinylmethyldimethoxysilane, vinylmethyldiethoxysilane, vinyltris(2-methoxyethoxy)silane, vinyltris(trimethylsiloxysilane) ) silane, 4-vinylphenyltrimethoxysilane, allyltrimethoxysilane, allyltriethoxysilane, 5-(triethoxysilyl)-2-norbornene; Styryl-based: p-styryltrimethoxysilane; Methacryl-based: 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyl
• the present invention provides a method for measuring glycated protein.
• the method for measuring glycated protein of the present invention is a method for measuring glycated protein in which glycated protein is detected by the reaction of fructosyl amino acid oxidase immobilized on a support.
• FIG. 3 shows the configuration of the glycated protein sensor 10, which is one embodiment of the present invention.
• the sensor 10 shown in FIG. 3 has an enzyme layer 12 containing fructosyl amino acid oxidase and protease, and a hydrogen peroxide detector 14 .
• Fructosyl amino acid oxidase and protease are cross-linked to the support bovine serum albumin with the cross-linking agent glutaraldehyde.
• Bovine serum albumin is also cross-linked to each other by glutaraldehyde.
• the enzyme layer 12 is laminated on the detection surface 16 side of the hydrogen peroxide detection unit 14 and bonded to the detection surface 16 of the hydrogen peroxide detection unit 14 with a silane coupling agent 18 .
• the glycated protein sensor of another embodiment has another layer in addition to the enzyme layer and the hydrogen peroxide detector.
• the other layer is, for example, a restricted permeation layer, and the restricted permeation layer may contain polycarbonate or an ion exchange resin.
• the restricted permeation layer may be provided outside the enzyme layer or between the enzyme layer and the hydrogen peroxide detector, and bonded by hydrogen bonding, ionic bonding, or the like.
• a cation exchange resin and/or an anion exchange resin are used.
• a cation exchange resin such as Nafion (registered trademark)
• an anion exchange resin such as polypyrrole
• the restricted permeation layer may comprise one, multiple or at least one type of ion exchange resin and may be configured with one, multiple or at least one type of layer.
• the glycated protein to be measured When the glycated protein to be measured is introduced, the glycated protein is degraded by the protease immobilized on the support, producing glycated peptide fragments and non-glycated peptide fragments. These peptide fragments are thought to diffuse in the enzyme layer 12, and when the saccharified peptide fragments react with the immobilized fructosyl amino acid oxidase, hydrogen peroxide is produced.
• the hydrogen peroxide detection unit 14 detects this hydrogen peroxide and outputs a signal related to its concentration.
• the hydrogen peroxide detector 14 is a hydrogen peroxide electrode
• the hydrogen peroxide is decomposed by the hydrogen peroxide electrode, and emitted electrons are detected as current.
• the glycated protein sensor 10 has a controller.
• the controller calculates the glycated protein concentration from the current detected at the hydrogen peroxide electrode.
• the controller can calculate the concentration of the glycated protein in the sample solution from a predetermined relationship between the concentration of the glycated protein and the current value generated by the hydrogen peroxide electrode.
• Fructosyl amino acid oxidase has high thermal stability when immobilized on bovine serum albumin, which is a support, and/or has fructosyl lysine-specific reactivity. It is possible to accurately calculate the concentration of glycated protein.
• FIG. 1 Another embodiment of the present invention is shown in FIG.
• a layer (fructosyl amino acid oxidase layer 22 ) containing a support on which fructosyl amino acid oxidase is immobilized is formed on the hydrogen peroxide detector 14 .
• a layer containing a protease-immobilized support (protease layer 24 ) is formed on the fructosyl amino acid oxidase layer 22 , that is, on the surface opposite to the hydrogen peroxide detector 14 . That is, the fructosyl amino acid oxidase layer 22 and the protease layer 24 are laminated in this order on the hydrogen peroxide detection section 14 .
• the glycated protein sensor of another embodiment has another layer in addition to the enzyme layer and the hydrogen peroxide detector.
• Other layers are, for example, the above-described restricted permeation layer, outside the fructosyl amino acid oxidase layer 22, between the fructosyl amino acid oxidase layer 22 and the protease layer 24, between the fructosyl amino acid oxidase layer 22 and the hydrogen peroxide detector. 14 and can be joined by hydrogen bonding, ionic bonding, or the like.
• the glycated protein to be measured is introduced, the glycated protein is decomposed by the proteases of the protease layer 24 to produce glycated peptide fragments and non-glycated peptide fragments. These peptide fragments are considered to permeate the fructosyl amino acid oxidase layer 22, and when the saccharified peptide fragments react with the immobilized fructosyl amino acid oxidase, hydrogen peroxide is generated, which is generated by the hydrogen peroxide detection unit 14. Detects hydrogen peroxide.
• the glycated protein sensor has a fructosyl amino acid oxidase layer but no protease immobilized on a support.
• a solution previously treated with protease may be introduced into the glycated protein sensor as a measurement target, and hydrogen peroxide produced in the fructosyl amino acid oxidase layer may be detected in the hydrogen peroxide detection unit.
• fructosyl amino acid oxidase used in this example.
• a gene containing a base sequence encoding the amino acid sequences shown in SEQ ID NOs: 1 to 5 and designed to add a histidine tag to the N-terminus of each inserted protein was introduced into an expression vector having a lactose operon.
• Escherichia coli BL21(DE3) strain (Novagen, Merck) was transformed with the expression vector.
• the fructosyl amino acid oxidases having the amino acid sequences shown in SEQ ID NOs: 1 to 5 produced by the E. coli are referred to as Examples 1 to 5, respectively.
• the target protein could be produced from Escherichia coli into which the base sequence encoding the fructosyl amino acid oxidase consisting of the amino acid sequences shown in SEQ ID NOs: 1-3 and 5 was introduced.
• Each E. coli was cultured with shaking in an LB liquid medium containing 50 mg/mL kanamycin (manufactured by Nacalai Tesque) for 2.5 hours. Furthermore, using LB liquid medium supplemented with isopropyl ⁇ -D-thiogalactopyranoside (hereinafter referred to as "IPTG”) to a final concentration of 0.5 mM and LB liquid medium without IPTG, Shaking culture was carried out overnight. After culturing, OD600 was measured to confirm growth of E. coli.
• IPTG isopropyl ⁇ -D-thiogalactopyranoside
• Cell lysis buffer (98 vol% HEPES buffer, 1 vol% 100 mg/mL lysozyme solution, 1 vol% 10% Triton (registered trademark) X- 100 solution containing 1% by volume of 0.5M Tris(2-carboxyethyl)phosphine and 0.2% by volume of 10U/ ⁇ L DpnI) was added so that the cell density was constant, and placed on ice. Incubated for 1 hour at The supernatant was collected by centrifugation, and HEPES buffer was added to the precipitate and mixed.
• sample buffer (2ME+) (X2) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to 8 ⁇ L of each of the supernatant and precipitate, mixed, and heat-treated at 95° C. for 10 minutes. It was applied to a gel for SDS-PAGE (Perfect NT Gel M, manufactured by DRC), electrophoresed at a constant voltage of 180 V, and stained using Quick CBB Plus (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). .
• FIG. 5 (A) is the result of electrophoresis of the supernatant, and (B) is the result of electrophoresis of the precipitate.
• M indicates a molecular weight marker, the number in each lane indicates the sequence number of the protein introduced into E. coli, "IPTG (-)” indicates E. coli cultured without adding IPTG, “IPTG (+ )” indicates Escherichia coli cultured with the addition of IPTG.
• a protein of about 48 to 53 kDa was present in the supernatant and precipitate of the cells treated with the IPTG-added cell lysate, and the recombinant fructosyl amino acid oxidase of Examples 1 to 3 and 5 was produced. was confirmed.
• the introduced protein was purified from the transformed E. coli.
• Each Escherichia coli was cultured using the LB medium described above, and cultured at 18°C for 20 hours after induction of expression with IPTG. .5)), disrupted the cells with an ultrasonicator, and centrifuged to obtain a cell lysate supernatant. After the cell lysate supernatant was filtered with a pore size of 0.45 ⁇ m, it was applied to HisTrap HP (GE Healthcare), washed with 6 times the column volume of buffer A, and further eluted with an elution buffer (0.25 M Elution was performed with 20 mM Tris-HCl (pH 7.5) containing sodium chloride and 0.5 M imidazole.
• the resulting eluate was dialyzed against 20 mM potassium phosphate buffer (pH 7.5) and sterilized through a filter with a pore size of 0.22 ⁇ m to obtain a sample.
• the protein concentration in the sample was measured using Protein Assay Kit I (manufactured by Bio-Rad). The volumes of samples containing Examples 1-5 and the protein concentrations in the samples are shown in Table 7.
• fructosyl amino acid oxidase activity of Examples 1-5 was examined.
• fructosyl amino acid oxidase FAOD-E manufactured by Kikkoman Corporation, Comparative Example 1
• Lucica registered trademark
• GA-L manufactured by Asahi Kasei Pharma Corporation
• SEQ ID NO: 26 A fructosyl amino acid oxidase having the amino acid sequence shown (Comparative Example 3) was used.
• SEQ ID NO: 26 is known as FAD-dependent oxidoreductase [Accession Number: KZZ92706] from Ascosphaera apis, and has 55-63% homology with the amino acid sequence of a known fructosyl amino acid oxidase.
• the fructosyl amino acid oxidase of Comparative Example 1 was purified in the same manner as in Examples 1 to 5 to obtain 10 mL of protein solution with a concentration of 15.8 mg/mL.
• the substrates used were fructosyllysine (F-Lys), fructosylvalylhistidine (F-Val-His), fructosylvaline (F-Val) and fructosylglycine (F-Gly).
• F-Lys fructosyllysine
• F-Val-His fructosylvalylhistidine
• F-Val fructosylvaline
• F-Gly fructosylglycine
• Comparative Example 2 240 ⁇ L of pretreatment solution (GAR-1) was added to the wells, and 6 ⁇ L of substrate was added. After reacting at 37°C for 5 minutes, 60 ⁇ L of enzyme solution (GAR-2) was added and further reacted at 37°C for 5 minutes. Immediately before the addition of the enzyme solution and 5 minutes after the addition, the absorbance was measured at a dominant wavelength of 546 nm and a sub-wavelength of 700 nm using purified water as a control, and the change in absorbance during that period was obtained.
• GAR-1 pretreatment solution
• substrate 60 ⁇ L of enzyme solution (GAR-2) was added and further reacted at 37°C for 5 minutes.
• the absorbance was measured at a dominant wavelength of 546 nm and a sub-wavelength of 700 nm using purified water as a control, and the change in absorbance during that period was obtained.
• fructosyl amino acid oxidase activity was confirmed when F-Lys was used as a substrate, so F-Val-His, F-Val, and F-Gly Activity against each was measured.
• Tables 8 and 9 show the activities of Examples and Comparative Examples against F-Lys and the specific activities against F-Val-His, F-Val, and F-Gly, respectively. Examples 1 to 5 were confirmed to have high substrate specificity for F-Lys.
• Fructosyl amino acid oxidase consisting of the amino acid shown in SEQ ID NO: 1 purified by the above method (Example 1) and, as a comparative example, FAOD-E (manufactured by Kikkoman Corporation, Comparative Example 1) were heated in a liquid phase. Stability and thermal stability in the immobilized state were verified.
• thermostability of the enzyme of Example 1 and that of Comparative Example 1 were compared in the liquid phase.
• 80 ⁇ L of HEPES buffer containing 1 U/mL of each enzyme (hereinafter referred to as “enzyme diluent”) was dispensed into tubes and heated at 30° C., 40° C., 50° C., and 55° C. on a heat block. Each temperature was maintained for 15 minutes.
• a glycated protein sensor (example) was produced.
• a platinum electrode was subjected to silane coupling treatment to introduce an amino group onto the electrode surface.
• bovine serum albumin (BSA) was added to the TES buffer and the enzyme from Example 1 above was added and stirred.
• BSA bovine serum albumin
• glutaraldehyde solution was added to the mixed solution of the enzyme and BSA, and the mixture was stirred. 1 ⁇ L of this solution was dropped onto a platinum electrode into which an amino group was introduced, and dried sufficiently to obtain the sensor of the example.
• the enzyme of Comparative Example 1 (FAOD-E, manufactured by Kikkoman Corporation) was used.
• HEPES buffer solution containing 50 ⁇ M F-Lys was dropped onto the sensors of Examples and Comparative Examples to obtain current output values.
• Each sensor was immersed in a HEPES buffer heated to 50° C., 65° C., or 80° C. and allowed to stand at that temperature for 20 minutes. After that, each sensor was allowed to stand at 4° C. for 30 minutes in order to eliminate the influence of residual heat.
• Power output values for F-Lys were obtained in the same manner as the measurements before temperature treatment. Taking the power output value before temperature treatment as 100%, the ratio of the power output value after temperature treatment to the power output value before temperature treatment was calculated as the residual activity.
• Fig. 6 shows the measurement results of thermal stability in the liquid phase
• Fig. 7 shows the measurement results of thermal stability in the immobilized state.
• the fructosyl amino acid oxidase of Example 1 exhibited almost the same residual activity as that of Comparative Example in the liquid phase.
• the enzyme when the enzyme was immobilized on the support, it exhibited a residual activity of 70% or more for F-Lys even after treatment at 65°C or 80°C for 20 minutes, which was superior to the comparative examples. rice field.
• fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 18 (hereinafter referred to as Example 6) and fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 19 (hereinafter referred to as Example 7). ) was used.
• a nucleotide sequence encoding each amino acid sequence was introduced into an expression vector pET28b (Novagen, Merck), E. coli BL21 (DE3) strain (Nippon Gene) was transformed, inoculated into LB medium supplemented with kanamycin, and incubated at 37°C. was cultured overnight. Furthermore, LB medium was added for preculture, and IPTG was added to a final concentration of 0.5 mM to induce expression overnight. The next day, pellets containing each enzyme were collected and frozen.
• the frozen pellet was suspended in equilibration buffer (20 mM Tris-HCl (pH 8.4), 250 mM NaCl, 20 mM Imidazole), Lysozyme and TCEP were added, and the suspension was further suspended. After adding Triton X-100 and suspending, sonication was performed, DNase I was added and shaken, and the supernatant was recovered by centrifugation. The resulting sample was applied to the column, purified with elution buffer (20 mM Tris-HCl (pH 8.4), 250 mM NaCl, 20 mM Imidazole), and washed with TES Buffer (10 mM TES, 150 mM NaCl, pH 7.0). dialyzed twice. Purification of the desired enzyme was confirmed by a microvolume spectrophotometer.
• a glycated protein sensor In order to clarify the thermal stability of these enzymes immobilized on the support, we created a glycated protein sensor.
• a platinum electrode was subjected to silane coupling treatment to introduce an amino group onto the electrode surface.
• bovine serum albumin (BSA) was added to the TES buffer and the above enzymes were added and stirred.
• BSA bovine serum albumin
• glutaraldehyde solution was added to the mixed solution of the enzyme and BSA, and the mixture was stirred. 1 ⁇ L of this solution was dropped onto a platinum electrode into which an amino group was introduced, and dried sufficiently to obtain a sensor.
• Example 6 shows that the residual activity after heat treatment at 65 ° C. in a state immobilized on a support is significantly higher.
• Example 3 it showed significantly higher residual activity after heat treatment at 50-65°C while immobilized on the support.
• Variant 1 in which lysines at positions 64, 84, 87, 128, 157, 166, 167, 394, and 397 of Example 1 are substituted with arginine (SEQ ID NO: 27), Example 1 112, 137, 152, 247, 254, 255, 267, 277, and variant 2 in which arginine was substituted for lysine at positions 438 (SEQ ID NO: 28), and Example 1 34th, 177th, 233rd, 234th, 297th, 309th, 413th, 424th, 430th, and 443rd lysines were substituted with arginine for variant 3 (SEQ ID NO: 29) as described above.
• Glycated protein sensors prepared in the same manner and immobilized with Example 1 and modified variants 1 to 3 were prepared.
• Fig. 9(A) The residual activity after heating at 50°C is shown in Fig. 9(A), and the residual activity after heating at 50°C and after heating at 60°C is shown in Fig. 9(B). All of the variants had a significantly reduced residual activity compared to Example 1. Therefore, the lysine substituted for arginine in variants 1 to 3 played an important role in the thermal stability of Example 1. It was suggested. Further, when comparing the residual activities among the variants, the residual activity of variant 3 was significantly lower than that of variants 1 and 2. Therefore, in particular, it was shown that the lysine substituted for arginine in variant 3 is more important for the thermostability of example 1.
• variant 4 a variant of Example 1 (hereinafter referred to as “variant 4") was produced.
• variant 4 in the amino acid sequence shown in SEQ ID NO: 1, (1) when the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 1 are aligned, the amino acid sequence shown in SEQ ID NO: 2 is lysine.
• the positions of amino acids other than lysine in the amino acid sequence shown in SEQ ID NO: 1 are substituted with lysine from the amino acids other than lysine, and (2) amino acids other than lysine in the amino acid sequence shown in SEQ ID NO: 2,
• a position that is lysine in the amino acid sequence shown in SEQ ID NO:1 introduced a substitution from lysine to the amino acid at that position in the amino acid sequence shown in SEQ ID NO:2.
• the amino acid substitutions of variant 4 are shown in Table 10, and its amino acid sequence is shown in SEQ ID NO:30.
• a Hepes buffer (10 mM Hepes + 150 mM NaCl, pH 8.0) containing 1 mg/mL FAOD (Examples 1, 2, 8 and variant 3) and BSA was prepared.
• a predetermined amount of glutaraldehyde was additionally added under the conditions for fixing FAOD to BSA.
• an enzyme solution containing FAOD fixed to BSA by the addition of glutaraldehyde and an enzyme solution containing FAOD not fixed to BSA without the addition of glutaraldehyde were obtained.
• the enzyme activity was measured after keeping the enzyme solution on ice and after heating it to 48° C. on a heat block and keeping it for 15 minutes. Taking the activity value of the enzyme solution kept on ice as 100, the ratio of the activity value obtained after treatment at 48°C was calculated as the residual activity.
• Variant 5 (SEQ ID NO: 31) in which lysine at positions 233, 234, and 297 of Example 1 was substituted with arginine
• variant 6 (SEQ ID NO: 31) in which lysine at positions 309 and 413 of Example 1 was substituted with arginine
• variant 7 (SEQ ID NO: 33) in which lysines at positions 424, 430, and 443 of Example 1 were replaced with arginine
• lysines at positions 34 and 177 of Example 1 were replaced with arginine.
• Each substituted variant 8 (SEQ ID NO:34) was generated in the same manner as described above.
• the residual activity of each FAOD in the liquid phase when BSA was fixed and when BSA was not fixed was measured by the same method as described above. In this experiment, the concentration of BSA in Hepes buffer was 2.64% (w/v).
• Table 12 shows the residual activity of each FAOD when BSA was not fixed, the residual activity when BSA was fixed, and the rate of change in residual activity.
• Variant 6 and variant 7 showed a negative rate of change in residual activity compared to Example 1 and other variants.
• Comparative Example 4 [Accession Number: BAD54824] is a known FAOD derived from Aspergillus oryzae, and the amino acid sequence of Comparative Example 4 (SEQ ID NO: 24) has a high homology of 95.1% to the amino acid sequence of Example 1. have sex.
• the amino acid corresponding to lysine at position 247 in Example 1 is arginine, and the amino acid corresponding to lysine at position 277 in Example 1 is asparagine. Therefore, the influence of the lysines at positions 247 and/or 277 in Example 1 on the thermal stability of Example 1 was analyzed.
• variant 9 SEQ ID NO: 35 in which arginine was substituted for lysine at positions 247 and 277 of Example 1 was prepared by the same method as described above.
• the residual activity of each FAOD after immobilization with BSA in the liquid phase was measured.
• the Bonferroni method after one-way ANOVA was used. The results are shown in Table 13. Since the residual activity of Example 1 was significantly higher than that of Comparative Example 4 and Variant 9, lysines at positions 247 and/or 277 in Example 1 are important for thermal stability during immobilization of FAOD. suggested to play an important role.
• Example 9 in the amino acid sequence of Comparative Example 5 shown in SEQ ID NO: 25, (1) when the amino acid sequence shown in SEQ ID NO: 25 and the amino acid sequence shown in SEQ ID NO: 1 were aligned, the amino acid sequence shown in SEQ ID NO: 1 was obtained. is lysine, and in the amino acid sequence shown in SEQ ID NO: 25, the amino acid other than lysine is substituted with lysine, and (2) in the amino acid sequence shown in SEQ ID NO: 25, A position that is an amino acid and is lysine in the amino acid sequence shown in SEQ ID NO:1 introduced a substitution from lysine to the amino acid at that position in the amino acid sequence shown in SEQ ID NO:25.
• the amino acid substitutions of FAOD of Example 9 are shown in Table 14, and its amino acid sequence is shown in SEQ ID NO:23.
• glycated protein sensor 10 glycated protein sensor 12 enzyme layer 14 hydrogen peroxide detector 16 detection surface 18 silane coupling agent 20 glycated protein sensor 22 fructosyl amino acid oxidase layer 24 protease layer

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Analytical Chemistry (AREA)
• Physics & Mathematics (AREA)
• Immunology (AREA)
• Biophysics (AREA)
• Medicinal Chemistry (AREA)
• Biomedical Technology (AREA)
• Enzymes And Modification Thereof (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (3)

Applications Claiming Priority (2)

Publications (1)

Family

ID=87472003

Family Applications (1)

Country Status (4)

Citations (10)

• 2023
  • 2023-01-23 JP JP2023576900A patent/JPWO2023145689A1/ja active Pending
  • 2023-01-23 WO PCT/JP2023/001930 patent/WO2023145689A1/ja not_active Ceased
  • 2023-01-23 EP EP23745039.0A patent/EP4471135A4/en active Pending
  • 2023-01-23 CN CN202380014997.3A patent/CN118369420A/zh active Pending

Patent Citations (10)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events