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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • 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 fructosyl amino acid oxidase, a gene encoding the amino acid sequence of fructosyl amino acid oxidase, an expression vector containing the gene, a glycated protein sensor having fructosyl amino acid oxidase immobilized on a support, a method for measuring glycated proteins that detects glycated proteins by the reaction of fructosyl amino acid oxidase immobilized on a support, and a method for producing fructosyl amino acid oxidase.
• Glycated proteins are measured as an indicator for diagnosing diabetes and for managing blood sugar control. 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, immunoassays, and enzyme methods.
• the enzymatic method for measuring glycated proteins involves, in the first step, decomposing proteins into amino acids or peptides using protease, and in the second step, treating those amino acids or peptides with fructosyl amino acid oxidase to generate hydrogen peroxide on glycated amino acids (hereinafter sometimes referred to as "glycated amino acids” or “fructosyl amino acids”) or peptides containing glycated amino acids (hereinafter sometimes referred to as "glycated peptides" or "fructosyl peptides”), and in the third step, converting the hydrogen peroxide into a color-developing reaction and measuring the absorbance, or detecting electrons released when hydrogen peroxide is decomposed at an electrode (see Patent Document 1).
• amadoriases that can be used to measure glycated hemoglobin accurately in samples containing glycated abnormal hemoglobin in response to various genotypes (see Patent Document 2), amadoriases obtained by substituting amino acids on a specific amino acid sequence so as to improve the specific activity against glycated substrates (see Patent Document 3), amadoriases obtained by substituting amino acids on a specific amino acid sequence so as to retain activity in the presence of surfactants (see Patent Documents 4 and 5), fructosyl amino acid oxidases obtained by substituting amino acids on a specific amino acid sequence so as to have high thermal stability (see Patent Documents 6 and 7), amadoriases that are not easily affected by oxygen concentration (see Patent Document 8), and a method for measuring fructosyl lysine using fructosyl amino acid oxidase from Asperg
• the problem that the present invention aims to solve is to provide a fructosyl amino acid oxidase that has physicochemical properties, such as high enzyme activity and high thermal stability, that contribute to the accuracy of measurements using a glycosylated protein sensor and the stability of the sensor.
• the inventors discovered a fructosyl amino acid oxidase with novel physicochemical properties from among genes with unknown functions, and further discovered that the physicochemical properties of the fructosyl amino acid oxidase could be improved by substituting specific amino acids on the fructosyl amino acid oxidase, thus arriving at the present invention.
• the present invention is a fructosyl amino acid oxidase having an amino acid sequence in which, when aligned with the amino acid sequence shown in SEQ ID NO:1, any amino acid corresponding to a position selected from the group consisting of positions 5, 58, 59, 98, 225, 277, 285, 355, and 440 in the amino acid sequence shown in SEQ ID NO:1 is substituted.
• the present invention may be a fructosyl amino acid oxidase having higher fructosyl amino acid oxidase activity and/or thermal stability than fructosyl amino acid oxidases having no amino acid substitutions, such as fructosyl amino acid oxidases having the amino acid sequence shown in SEQ ID NO: 1 or 199. More specifically, the fructosyl amino acid oxidase has fructosyl amino acid oxidase activity and/or thermal stability that are 4% or more higher than fructosyl amino acid oxidases having no amino acid substitutions, such as fructosyl amino acid oxidases having the amino acid sequence shown in SEQ ID NO: 1 or 199.
• the present invention may be a fructosyl amino acid oxidase having a higher activity and/or thermal stability when immobilized on a support than a fructosyl amino acid oxidase in which the amino acid is not substituted, such as a fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 1 or 199.
• the fructosyl amino acid oxidase has a higher activity after heat treatment than that of a fructosyl amino acid oxidase having no amino acid substitutions, such as a fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 1 or 199.
• the heat treatment conditions are, for example, 45°C or 48°C for 15 minutes.
• the fructosyl amino acid oxidase may have a higher thermal stability against heat treatment than a fructosyl amino acid oxidase having no amino acid substitution, such as a fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 1 or 199.
• the heat treatment conditions are, for example, 45°C or 48°C for 15 minutes.
• the fructosyl amino acid oxidase may have a reactivity specific to fructosyl lysine.
• the fructosyl amino acid oxidase may have a reactivity to fructosyl valine, a peptide containing fructosyl valine, and fructosyl glycine of 20 or less, where the reactivity to fructosyl lysine is taken as 100.
• Another aspect of the present invention is a fructosyl amino acid oxidase having an amino acid sequence showing 75% or more homology to the amino acid sequence shown in SEQ ID NO: 1.
• the amino acid sequence of the fructosyl amino acid oxidase of the present invention may be an amino acid sequence that satisfies at least any of the following (1) to (9).
• the amino acid corresponding to the 5th position in the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid corresponding to the 58th position in the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid corresponding to the 59th position in the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, (4)
• the amino acid corresponding to the 98th position of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid corresponding to the 225th position of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid corresponding to position 277 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid corresponding to position 285 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid corresponding to position 355 of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid corresponding to position 440 of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• another aspect of the present invention is a fructosyl amino acid oxidase having an amino acid sequence showing 79% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and when aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 58 in the amino acid sequence shown in SEQ ID NO: 1 is any one of histidine, serine, threonine, glycine, alanine, valine, leucine, isoleucine, and phenylalanine, or any one of phenylalanine, alanine, and isoleucine.
• another aspect of the present invention is a fructosyl amino acid oxidase having an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 198 and SEQ ID NOs: 200 to 218.
• another aspect of the present invention may be a fructosyl amino acid oxidase having an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted, substituted, added, and/or inserted in the amino acid sequence.
• the present invention provides a gene encoding the amino acid sequence of the above-mentioned fructosyl amino acid oxidase, and an expression vector containing the gene.
• the present invention further provides a glycated protein sensor comprising fructosyl amino acid oxidase and a support.
• the fructosyl amino acid oxidase can be immobilized on the support by crosslinking with an amine-reactive crosslinker.
• the output value of the sensor after being left standing at a temperature of 65°C or 80°C for 20 minutes can be 60% or more of the output value of the sensor before being left standing.
• the output value of the sensor after being left standing at a temperature of 75°C and a relative humidity of 60% for 1 or 2 days can be 40%, 50%, 60%, or 70% or more of the output value of the sensor before being left standing.
• the present invention further provides a method for measuring glycated proteins by detecting glycated proteins by the reaction of fructosyl amino acid oxidase, and a method for producing fructosyl amino acid oxidase, comprising an amino acid substitution step of substituting any amino acid corresponding to a position selected from the group consisting of positions 5, 58, 225, 277, and 440 in the amino acid sequence shown in SEQ ID NO: 1 when the amino acid sequence of fructosyl amino acid oxidase is aligned with the amino acid sequence shown in SEQ ID NO: 1.
• the amino acid sequence of fructosyl amino acid oxidase before amino acid substitution used in the production method of the present invention may be an amino acid sequence that shows high homology to the amino acid sequence shown in SEQ ID NO: 1, for example, 50% or more, 60% or more, 70% or more, 75% or more, or 79% or more.
• the fructosyl amino acid oxidase of the present invention is heat stable when immobilized on a support and/or when not immobilized on a support.
• the fact that the fructosyl amino acid oxidase of the present invention is heat stable when immobilized on a support is advantageous in that the long-term stability of a glycated protein sensor comprising an immobilized enzyme is improved.
• the glycated protein sensor is capable of obtaining highly reliable measurement results over a long period of time, since the decrease in enzyme activity during storage is suppressed.
• the present invention it is possible to obtain a fructosyl amino acid oxidase having higher enzymatic activity and thermal stability than a wild-type fructosyl amino acid oxidase.
• the production method of the present invention can produce a variant having higher activity and/or thermal stability from a known fructosyl amino acid oxidase.
• FIG. 1 is a diagram showing the results of an experiment evaluating the thermal stability of a sensor comprising fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO:1.
• FIG. 1 shows the output value (left) and the rate of change in the output value (right) of a sensor equipped with fructosyl amino acid oxidase (wild-type) having the amino acid sequence shown in SEQ ID NO: 1, and a sensor equipped with fructosyl amino acid oxidase (M58F modified form) having the amino acid sequence shown in SEQ ID NO: 38.
• Statistical significance between the two groups was calculated using a t-test. In the figure, "***" indicates that the p-value was less than 0.001, "**” indicates that the p-value was less than 0.01, and "n.s.” indicates that no statistically significant difference was confirmed.
• the fructosyl amino acid oxidase of the present invention has high enzymatic activity and high thermostability. Moreover, the fructosyl amino acid oxidase of one aspect of the present invention has high thermostability when immobilized on a support.
• the present invention also provides a gene encoding the amino acid sequence of fructosyl amino acid oxidase, and an expression vector containing said gene.
• the present invention also provides a glycated protein sensor comprising a support and fructosyl amino acid oxidase immobilized on the support, a method for measuring glycated proteins that detects glycated proteins by the reaction of fructosyl amino acid oxidase immobilized on the support, and a method for producing fructosyl amino acid oxidase.
• a glycated protein sensor comprising a support and fructosyl amino acid oxidase immobilized on the support, a method for measuring glycated proteins that detects glycated proteins by the reaction of fructosyl amino acid oxidase immobilized on the support, and a method for producing fructosyl amino acid oxidase.
• Fructosyl amino acid oxidase is an enzyme that acts on amino acids whose ⁇ -amino group and/or ⁇ -amino group are glycated, or on peptides containing such amino acids, and produces hydrogen peroxide in the process of deglycosylating the glycated amino acids.
• Fructosyl amino acid oxidase (hereinafter sometimes referred to as "FAOD”) is also called amadoriase, ketoamine oxidase, or fructosyl amine oxidase.
• Fructosyl amino acid oxidase includes fructosyl peptide oxidase (hereinafter sometimes referred to as "FPOD" or "FPOX”), which acts on glycated peptides.
• Fructosyl amino acid oxidase uses glycated amino acids and/or glycated peptides as substrates.
• glycated amino acids include ⁇ -fructosyl lysine (sometimes called “fructosyl lysine”), ⁇ -fructosyl valine (sometimes called “fructosyl valine”), ⁇ -fructosyl glycine (sometimes called “fructosyl glycine”), ⁇ -fructosyl histidine (sometimes called “fructosyl histidine"), ⁇ -fructosyl leucine (sometimes called “fructosyl leucine”), and ⁇ -fructosyl serine (sometimes called “fructosyl serine”)
• glycosylated peptides include peptides consisting of 2 to 10 amino acids, preferably 2 to 6 amino acids, and more preferably 2 to 3 amino acids, and including one or more glycosylated amino acids.
• ⁇ -fructosyl valyl histidine (sometimes called “fructosyl valyl histidine”) is included.
• the fructosyl amino acid oxidase of the present invention has the following physicochemical properties: It has an optimal pH range of pH 7 to 9, an active pH range of pH 5 to 9, an active temperature range of 20 to 80°C, and is soluble in a buffer solution.
• the buffer solution include Tris hydrochloride buffer and phosphate buffered saline (PBS).
• the fructosyl amino acid oxidase of the present invention may specifically react with a specific glycated amino acid or a peptide containing the amino acid.
• the fructosyl amino acid oxidase is highly specific to fructosyl lysine. High specificity to fructosyl lysine means that when the reactivity to fructosyl lysine or a peptide containing fructosyl lysine is taken as 100, the reactivity to other glycated amino acids or peptides containing other glycated amino acids is 20 or less.
• the reactivity to other glycated amino acids or peptides containing other glycated amino acids is preferably 10 or less, more preferably 5 or less, and even more preferably 1 or less.
• the other glycated amino acid refers to, for example, one or more selected from fructosyl valine, fructosyl glycine, fructosyl histidine, fructosyl leucine, and fructosyl serine.
• Fructosyl amino acid oxidases are classified into three groups based on substrate specificity.
• Fructosyl amino acid oxidases belonging to group 1 are highly specific to amino acids whose ⁇ -amino group is glycated and/or peptides containing said amino acids.
• Fructosyl amino acid oxidases belonging to group 2 are highly specific to amino acids whose ⁇ -amino group is glycated and/or peptides containing said amino acids.
• Fructosyl amino acid oxidases belonging to group 3 are highly specific to amino acids whose ⁇ -amino group is glycated and/or peptides containing said amino acids, as well as amino acids whose ⁇ -amino group is glycated and/or peptides containing said amino acids.
• the fructosyl amino acid oxidase of the present invention may belong to any of the groups.
• the fructosyl amino acid oxidase is highly specific to fructosyl valyl histidine.
• High specificity to fructosyl valyl histidine means that, when the reactivity to fructosyl valyl histidine is taken as 100, the reactivity to other glycated amino acids or other glycated peptides is 20 or less.
• the reactivity to other glycated amino acids or other fructosyl peptides is preferably 10 or less, more preferably 5 or less, and even more preferably 1 or less.
• the other glycated amino acids refer to, for example, one or more selected from fructosyl valine, fructosyl lysine, fructosyl glycine, fructosyl histidine, fructosyl leucine, and fructosyl serine, and the other fructosyl peptides refer to peptides containing glycated amino acids other than fructosyl valyl histidine.
• the fructosyl amino acid oxidase of the present invention may specifically react with a specific glycated amino acid or a peptide containing the amino acid.
• the fructosyl amino acid oxidase is highly specific to fructosyl valine. High specificity to fructosyl valine means that, when the reactivity to fructosyl valine or a peptide containing fructosyl valine is taken as 100, the reactivity to other glycated amino acids or peptides containing other glycated amino acids is 20 or less.
• the reactivity to other glycated amino acids or peptides containing other glycated amino acids is preferably 10 or less, more preferably 5 or less, and even more preferably 1 or less.
• the other glycated amino acid refers to, for example, one or more selected from fructosyl lysine, fructosyl glycine, fructosyl histidine, fructosyl leucine, and fructosyl serine.
• the inventors discovered a novel fructosyl amino acid oxidase from among genes with unknown functions. Among them, a fructosyl amino acid oxidase was obtained that exhibited significantly high thermal stability when immobilized on a support and/or when not immobilized on a support, and therefore a modified fructosyl amino acid oxidase was created based on the amino acid sequence of the fructosyl amino acid oxidase.
• the amino acid sequence shown in SEQ ID NO:1 is the amino acid sequence of fructosyl amino acid oxidase derived from Aspergillus pseudotamarii of the genus Aspergillus. Until now, it was predicted to be a type of FAD-dependent oxidoreductase based on sequence homology [Accession Number: XP_031920077], but it was not known that a protein containing the amino acid sequence shown in SEQ ID NO:1 could function as a fructosyl amino acid oxidase.
• the present inventors have found that a fructosyl amino acid oxidase containing the amino acid sequence shown in SEQ ID NO:1 has remarkably high thermal stability when immobilized on a support, and has extremely high substrate specificity for a specific glycated amino acid and/or glycated peptide.
• the present invention further provides a fructosyl amino acid oxidase comprising an amino acid sequence having high homology to SEQ ID NO: 1, in which one or more amino acids corresponding to positions selected from the group consisting of positions 5, 58, 225, 277, and 440 in the amino acid sequence shown in SEQ ID NO: 1 have been substituted when aligned with the amino acid sequence shown in SEQ ID NO: 1.
• fructosyl amino acid oxidases consisting of known amino acid sequences are excluded from the fructosyl amino acid oxidases of the present invention even if they satisfy the above conditions.
• amino acid sequence that is highly homologous to SEQ ID NO:1 is one that has a homology of 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 51% or more, 52% or more, 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% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more with respect to the amino acid sequence of SEQ ID NO:1.
• the amino acid at position 5 of the amino acid sequence shown in SEQ ID NO:1 is asparagine.
• the amino acid sequence of the fructosyl amino acid oxidase of one embodiment of the present invention is an amino acid sequence in which, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 5 of SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid sequence of the fructosyl amino acid oxidase shown in SEQ ID NO:199 is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 5 of SEQ ID NO:1 is isoleucine, but the amino acid sequence shown in SEQ ID NO:199 is excluded from the amino acid sequence of the fructosyl amino acid oxidase of the present invention.
• BAD54824 and the amino acid sequence of the fructosyl amino acid oxidase disclosed in Patent Document 9 is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 5 of SEQ ID NO:1 is lysine, but the amino acid sequences of these known fructosyl amino acid oxidases are excluded from the amino acid sequence of the fructosyl amino acid oxidase of the present invention.
• the amino acid at position 58 in the amino acid sequence shown in SEQ ID NO:1 is methionine.
• the amino acid sequence of the fructosyl amino acid oxidase of one embodiment of the present invention is an amino acid sequence in which, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 58 in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid at position 59 in the amino acid sequence shown in SEQ ID NO:1 is glutamic acid.
• the amino acid sequence of the fructosyl amino acid oxidase of one embodiment of the present invention is an amino acid sequence in which, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 59 in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid at position 98 in the amino acid sequence shown in SEQ ID NO:1 is glutamic acid.
• the amino acid sequence of the fructosyl amino acid oxidase of one embodiment of the present invention is an amino acid sequence in which, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 98 in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid at position 225 of the amino acid sequence shown in SEQ ID NO:1 is glycine.
• the amino acid sequence of the fructosyl amino acid oxidase of one embodiment of the present invention is an amino acid sequence in which, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 225 of SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid at position 277 in the amino acid sequence shown in SEQ ID NO:1 is lysine.
• the amino acid sequence of the fructosyl amino acid oxidase of one embodiment of the present invention is an amino acid sequence in which, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 277 in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid at position 285 of the amino acid sequence shown in SEQ ID NO:1 is glutamic acid.
• the amino acid sequence of the fructosyl amino acid oxidase of one embodiment of the present invention is an amino acid sequence in which, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 285 of SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid at position 355 of the amino acid sequence shown in SEQ ID NO:1 is aspartic acid.
• the amino acid sequence of the fructosyl amino acid oxidase of one embodiment of the present invention is an amino acid sequence in which, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 355 of SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• the amino acid at position 440 in the amino acid sequence shown in SEQ ID NO:1 is glycine.
• the amino acid sequence of the fructosyl amino acid oxidase of one embodiment of the present invention is an amino acid sequence in which, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO:1, the amino acid corresponding to position 440 in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• a fructosyl amino acid oxidase having an amino acid sequence with multiple amino acid substitutions as described above.
• fructosyl amino acid oxidase of the present invention may be a fructosyl amino acid oxidase that includes any of the following amino acid sequences or that consists of any of the following amino acid sequences.
• the fructosyl amino acid oxidase may be a fructosyl amino acid oxidase that consists of any of the following amino acid sequences.
• amino acid sequence of the fructosyl amino acid oxidase of the present invention is shown below.
• the methionine which is the amino acid at position 58 of the amino acid sequence shown in SEQ ID NO: 199, is replaced with any amino acid selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
• amino acid sequence shown in SEQ ID NO:199 is fructosyl amino acid oxidase derived from Aspergillus fumigatus [Accession Number: AAB88209], and is also called "amadoriase I".
• the amino acid sequence shown in SEQ ID NO:199 was found to have 79.2% amino acid homology with the amino acid sequence shown in SEQ ID NO:1.
• the amino acid sequences shown in SEQ ID NO:200 to 218 obtained by substituting one amino acid in the amino acid sequence shown in SEQ ID NO:199 were found to have 79.0% amino acid homology with the amino acid sequence shown in SEQ ID NO:1.
• Another embodiment of the present invention is a fructosyl amino acid oxidase having an amino acid sequence in which one or several amino acids have been modified or mutated, or deleted, substituted, added and/or inserted in the amino acid sequence shown in any of the above SEQ ID NOs, and having physicochemical properties such as high thermostability and high substrate specificity for specific glycated amino acids or glycated peptides.
• "one or several amino acids” refers to 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1 or 2 amino acids.
• the present invention also provides a gene comprising a base sequence encoding the above-mentioned amino acid sequence.
• the gene may be DNA or RNA, and may be complementary DNA (cDNA).
• the present invention further provides an expression vector comprising a gene comprising a base sequence encoding the following amino acid sequence:
• the fructosyl amino acid oxidase of the present invention may be a recombinant protein obtained by introducing a nucleic acid containing a base sequence encoding the fructosyl amino acid oxidase of the present invention into a host such as Escherichia coli, yeast, mammalian cells, or insect cells, and expressing the nucleic acid.
• the recombinant protein may also be a recombinant protein synthesized in a cell-free protein synthesis system.
• the fructosyl amino acid oxidase comprises the amino acid sequence described above.
• an amino acid sequence that functions as a tag may be added to the N-terminus and/or C-terminus to facilitate purification and detection of the enzyme.
• tags include His tag, Myc tag, HA tag, and FLAG tag.
• the fructosyl amino acid oxidase of the present invention has high activity against a substrate when immobilized on a support and/or when not immobilized on a support.
• the fructosyl amino acid oxidase obtained by the above-mentioned amino acid substitution has higher activity against a substrate than a fructosyl amino acid oxidase consisting of an amino acid sequence in which no amino acids are substituted.
• the fructosyl amino acid oxidase of the present invention may have activity improved by 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to the fructosyl amino acid oxidase before the amino acid substitution. These values are rounded off to one decimal place.
• the activity improvement rate of the fructosyl amino acid oxidase of the present invention compared to the wild type can be calculated, for example, by the following formula.
• the calculated activity improvement rate will be a positive value if the activity is improved by the amino acid substitution, and will be a negative value if the activity is decreased by the amino acid substitution.
• fructosyl amino acid oxidase of the present invention is a fructosyl amino acid oxidase whose activity measured after heat treatment of the fructosyl amino acid oxidase immobilized on a support and/or not immobilized on a support is improved compared to that of the wild type.
• the fructosyl amino acid oxidase of the present invention may have an activity after heat treatment that is improved by 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to the fructosyl amino acid oxidase before amino acid substitution. These values are rounded off to one decimal place (the same applies below).
• the fructosyl amino acid oxidase of the present invention is heat stable when immobilized on a support and/or when not immobilized on a support.
• the fructosyl amino acid oxidase obtained by the above-mentioned amino acid substitution has higher heat stability than a fructosyl amino acid oxidase having an amino acid sequence in which no amino acids are substituted.
• the fructosyl amino acid oxidase of the present invention may have heat stability improved by 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more compared to the fructosyl amino acid oxidase before the amino acid substitution. These values are rounded off to one decimal place (the same applies below).
• Fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO:1 is heat stable when immobilized on a support.
• a fructosyl amino acid oxidase that is heat stable when immobilized on a support is one that has high residual activity after heat treatment when immobilized on a support, and an example of such a fructosyl amino acid oxidase is one that has a residual activity of 70% or more after treatment at 65°C.
• the treatment temperature can be 45°C, 48°C, 50°C, 55°C, 60°C, or 65°C.
• the treatment time can be 10 to 30 minutes, preferably 15 to 25 minutes, and most preferably 20 minutes.
• the residual activity after heat treatment can be 30%, 40%, 50%, 60%, or 70% or more.
• the fructosyl amino acid oxidase of one embodiment of the present invention is included in a glycated protein sensor.
• a glycated protein sensor includes fructosyl amino acid oxidase immobilized on a support.
• the fructosyl amino acid oxidase included in the glycated protein sensor has extremely high residual activity after the protein sensor is heat-treated, so that the output value of the sensor is maintained high even when the sensor is placed in a high-temperature environment.
• the output value of the sensor after being left standing at a temperature of 65°C or 80°C for 20 minutes can be 60% or more of the output value of the sensor before being left standing.
• the output value of the sensor after being left standing at a temperature of 75°C and a relative humidity of 60% for 1 day, 2 days, or 3 days can be 40%, 50%, 60%, or 70% or more of the output value of the sensor before being left standing.
• fructosyl amino acid oxidase that is heat stable when immobilized on a support
• a fructosyl amino acid oxidase whose residual activity after heat treatment when immobilized on a support is higher than the residual activity after treatment under the same conditions when not immobilized on a support.
• the treatment temperature is 45°C, 48°C, 50°C, 55°C, 60°C, or 65°C.
• the treatment time is 10 to 30 minutes, preferably 15 to 25 minutes, and most preferably 20 minutes.
• the support on which fructosyl amino acid oxidase is immobilized is heated by known means such as an electric oven or a thermostatic 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.
• the fructosyl amino acid oxidase of the present invention obtained by substituting amino acids in the amino acid sequence shown in SEQ ID NO:1 has improved thermal stability compared to the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO:1.
• the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO:1 has extremely high thermal stability compared to other FAODs. Therefore, even if the improvement in thermal stability due to amino acid substitution is only a few percent, the effect can be said to be significant.
• thermostability specifically refers to thermostability with respect to heat treatment at 45°C or 48°C for 15 minutes.
• the thermostability of fructosyl amino acid oxidase can be evaluated by any method, but one example is a method in which the activity of heat-treated FAOD and the activity of non-heat-treated FAOD are measured, and the percentage of enzyme activity remaining after heat treatment is calculated as the residual activity.
• the rate of improvement in thermal stability due to amino acid substitution can be calculated using the following formula.
• the calculated rate of improvement in thermal stability will be a positive value if the amino acid substitution improves thermal stability, and a negative value if the amino acid substitution reduces thermal stability.
• the fructosyl amino acid oxidase of one embodiment of the present invention is stable to heat when immobilized on a support.
• means for immobilizing an enzyme such as FAOD on a support include covalent bonding, physical adsorption, ionic bonding, cross-linking, entrapment, and biochemical specific binding.
• an immobilization method that does not inactivate the enzyme may be selected, and multiple immobilization methods may be used in combination.
• the FAOD is immobilized on the support by crosslinking with a crosslinking agent, either alone or in a state where it is bound to a carrier.
• the support is a protein other than the FAOD
• the FAOD is immobilized on the support by mixing the FAOD with the support and then using a crosslinking agent such as glutaraldehyde or an isocyanate derivative.
• proteins include albumins such as bovine serum albumin (BSA), collagen, gelatin, etc.
• the crosslinking agent used is a substance that crosslinks between molecules or within molecules, and specific examples include glutaraldehyde, isocyanate derivatives, formaldehyde, glyoxal, malondialdehyde, and succinaldehyde. Among these, amine-reactive crosslinking agents are preferably used. Amine-reactive crosslinking agents react with the amino groups of proteins to crosslink the proteins, and specific examples include glutaraldehyde, formaldehyde, N-hydroxyesters, amide esters, and imide esters.
• the amine-reactive crosslinking agent reacts with the amino groups present on the surface of the fructosyl amino acid oxidase used in the present invention to form crosslinks, so that the fructosyl amino acid oxidase becomes stable against heat when immobilized on the support.
• the support may be a synthetic polymer, a resin, an inorganic material, a polysaccharide, a mineral, a clay, etc.
• the means for fixing FAOD to a support include fixing FAOD to a support made of fluororesin, ion exchange resin, polyvinyl alcohol resin, hydraulic resin, photocurable resin, solid polymer electrolyte, polyion complex, urethane, etc.; fixing FAOD to the support made of charcoal, bone charcoal, silica gel, glass, zeolite, celite, alumina, titanium oxide, ceramic, hydroxyapatite, etc.
• the support may also be a carbon material and/or beads.
• the carbon material may be carbon nanotubes, fullerenes, graphene, etc.
• the beads may be carbon microparticles (carbon beads), silica (SiO 2 ) microparticles (silica beads), or beads made of polysaccharides such as chitin, chitosan, or alginic acid.
• the beads may also contain metal microparticles or magnetizable substances, and may be magnetic beads.
• the beads may have an average particle size of 10 nm or more, or 200 nm or less.
• the FAOD may be immobilized by being crosslinked to the beads.
• 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.
• the enzyme includes at least fructosyl amino acid oxidase.
• the glycated protein sensor of the present invention may be a glycated protein sensor that calculates the amount of glycated protein contained in a test sample, i.e., the concentration of glycated protein in the test sample.
• the fructosyl amino acid oxidase contained in the sensor of the present invention is stable to heat when immobilized on a support, and is therefore characterized by the fact that it can provide reliable measurement results over a long period of time.
• the sensor of the present invention was left to stand at a temperature of 65°C or 80°C for 20 minutes, it maintained an output value of 70% or more of the value before standing.
• the sensor of the present invention was left to stand for one day under conditions of a temperature of 75°C and a relative humidity of 60%, it maintained an output value of 48% of the value before standing, and when the sensor was left to stand for two days, it maintained an output value of 29% of the value before standing.
• 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 unit.
• the detection unit detects hydrogen peroxide generated from glycated amino acids and/or glycated peptides by fructosyl amino acid oxidase.
• the glycated protein sensor is capable of calculating the amount (concentration) of glycated proteins 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.
• the hydrogen peroxide electrode detects electrons released when hydrogen peroxide is decomposed into oxygen as a current, and 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 or light intensity.
• hydrogen peroxide is quantified by detecting a color reaction in the presence of peroxidase and an oxidative coloring dye, or the luminescence intensity of luminol.
• the detection unit is an electrochemiluminescence detection unit, and a gold electrode, a platinum electrode, or an indium tin oxide transparent electrode (ITO electrode) is used. Hydrogen peroxide is detected by measuring the luminescence produced by a luminescent reagent such as luminol.
• the detection unit may be a hydrogen peroxide detection unit using a different method.
• the test sample for the glycated protein sensor of the present invention may be a solution.
• the solution may be a body fluid, a solution derived from a body fluid, or a dilution of a body fluid.
• the solution may be a solution that is not a body fluid (non-body fluid-derived), or a mixture of a body fluid or a solution derived from a body fluid and a solution derived from a non-body fluid.
• the solution may be a solution used for sample measurement, or a solution used for calibration measurement.
• the solution may be a standard solution or a calibration solution.
• the solution may contain a buffer solution.
• the body fluid may be blood, serum, plasma, lymphatic fluid, tissue fluid such as interstitial fluid, intercellular fluid, interstitial fluid, body cavity fluid, serous cavity fluid, pleural fluid, peritoneal fluid, pericardial fluid, cerebrospinal fluid, joint fluid, aqueous humor.
• the body fluid may be digestive fluid such as saliva, gastric juice, bile, pancreatic juice, intestinal fluid, sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, milk.
• the body fluid may be an animal body fluid or a human body fluid.
• the body fluid may be a liquid in a food containing animal-derived protein (e.g., milk or dairy products).
• the body fluid may be a plant body fluid, a plant biofluid, or a liquid derived from a plant.
• the body fluid may be a plant juice, nectar, or sap.
• the solution may contain a substance to be measured.
• the solution may be tears, and the substance to be measured may be albumin or glycoalbumin contained in the tears.
• the substance to be measured may be albumin, glycoalbumin, hemoglobin, or glycohemoglobin in blood, serum, or plasma, albumin or glycoalbumin in interstitial fluid, albumin or glycoalbumin in urine, or albumin or glycoalbumin in saliva.
• 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 specific examples include glycated albumin and glycated hemoglobin contained in blood.
• a protease immobilized on a support may also be included.
• Protease is a general term for peptide bond hydrolases that hydrolyze and catabolize proteins and polypeptides.
• Protease may be an enzyme that breaks down proteins into peptide fragments.
• the action of the protease may produce one or more selected from the group consisting of glycosylated amino acids, peptide fragments containing glycosylated amino acids, non-glycated amino acids, and peptide fragments not containing glycosylated amino acids.
• Fructosyl amino acid oxidase may react with glycosylated amino acids or peptide fragments containing glycosylated amino acids to produce hydrogen peroxide.
• the support to which the enzyme is immobilized forms a support layer, such as a thin film layer, and is arranged by being laminated against the detection surface of the detection unit.
• a bonding agent such as a silane coupling agent, is used to arrange the support on or near the detection surface of the detection unit.
• a bonding layer is formed between the layered support and the detection unit, thereby bonding the support and the detection surface.
• the present invention further provides a method for measuring glycated proteins.
• the method for measuring glycated proteins of the present invention is a method for measuring glycated proteins that detects glycated proteins by the reaction of fructosyl amino acid oxidase immobilized on a support.
• the method for measuring glycated proteins of the present invention may be a non-diagnostic and/or diagnostic method.
• the present invention further provides a method for producing fructosyl amino acid oxidase.
• the method for producing fructosyl amino acid oxidase of the present invention includes an amino acid substitution step of substituting any amino acid corresponding to a position selected from the group consisting of positions 5, 58, 225, 277, and 440 in the amino acid sequence shown in SEQ ID NO: 1 when the amino acid sequence of fructosyl amino acid oxidase is aligned with the amino acid sequence shown in SEQ ID NO: 1.
• the activity and/or thermal stability of the fructosyl amino acid oxidase produced by this production method, when immobilized on a support and/or when not immobilized on a support, is higher than the activity and/or thermal stability of fructosyl amino acid oxidase in which the amino acid is not substituted.
• amino acid sequence of the fructosyl amino acid oxidase used is one that is highly homologous to the amino acid sequence shown in SEQ ID NO: 1.
• an amino acid sequence that is highly homologous to SEQ ID NO: 1 is one that is 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 51% or more, 52% or more, 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% or more, 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, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more or more
• NCBI National Center for Biotechnology Information
• a glycated protein sensor was produced using wild-type or comparative enzymes.
• a platinum electrode was subjected to a silane coupling treatment to introduce amino groups onto the electrode surface.
• bovine serum albumin (BSA) was added to the TES buffer solution, and each enzyme was added and stirred.
• BSA bovine serum albumin
• glutaraldehyde solution was added to the mixture of enzymes and BSA, and stirred. 1 ⁇ L of this solution was dropped onto the platinum electrode with the amino groups introduced, and it was thoroughly dried to obtain a sensor.
• HEPES buffer containing 50 ⁇ M F-Lys 200 ⁇ L of HEPES buffer containing 50 ⁇ M F-Lys was dropped onto each sensor to obtain a current output value.
• Each sensor was immersed in HEPES buffer heated to 50°C, 65°C, or 80°C and left to stand at that temperature for 20 minutes. Then, to eliminate the effects of residual heat, each sensor was left to stand at 4°C for 30 minutes.
• the output value for F-Lys was obtained in the same manner as the measurement before temperature treatment.
• the output value before temperature treatment was set to 100%, and the ratio of the output value after temperature treatment to the output value before temperature treatment was calculated as the rate of change in the output value.
• Fructosyl lysine F-Lys
• fructosyl valyl histidine F-Val-His
• fructosyl valine F-Val
• fructosyl glycine F-Gly
• the wild-type and comparative example have fructosyl amino acid oxidase activity when F-Lys is used as a substrate
• the activity for each of F-Val-His, F-Val, and F-Gly was measured under the same conditions as for F-Lys.
• the specific activities of the wild-type and comparative example for each of F-Lys, F-Val-His, F-Val, and F-Gly are shown in the table below.
• the reactivity of the wild-type fructosyl amino acid oxidase (SEQ ID NO: 1) for F-Val-His, F-Val, and F-Gly was all less than 1, assuming that the reactivity for F-Lys is 100. It was confirmed that the wild-type fructosyl amino acid oxidase has F-Lys-specific reactivity.
• the detailed method for producing the fructosyl amino acid oxidase used in this example is described below.
• a gene containing a base sequence encoding the amino acid sequence described in this specification 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.
• the expression vector was used to transform E. coli BL21 (DE3) Derived strain (manufactured by Nippon Gene Co., Ltd.).
• Transformed E. coli was cultured for 2 hours with shaking in LB liquid medium containing 50 ⁇ g/mL kanamycin (Nacalai Tesque). These E. coli were then cultured for 20 hours at 16°C in LB liquid medium containing isopropyl ⁇ -D-thiogalactopyranoside (hereinafter referred to as "IPTG") at a final concentration of 0.1 mM.
• IPTG isopropyl ⁇ -D-thiogalactopyranoside
• the collected E. coli was suspended in buffer A (20 mM Tris-HCl (pH 7.5) containing 0.25 M sodium chloride and 20 mM imidazole), disrupted with an ultrasonic disrupter, and centrifuged to obtain a cell disruption supernatant.
• the cell lysate supernatant was filtered through a 0.45 ⁇ m pore size filter and then applied to EconoFit Nuvia IMAC Columns, Ni-charged (BIO-RAD), washed with 7 column volumes of buffer A, and then eluted with elution buffer (20 mM Tris-HCl (pH 7.5) containing 0.25 M sodium chloride and 0.5 M imidazole).
• the resulting eluate was dialyzed against 10 mM sodium phosphate buffer (pH 8.4) containing 150 mM sodium chloride and 50% by volume glycerol to obtain a sample.
• the protein concentration in the sample was measured using absorbance at 280 nm.
• Examples The activity and thermal stability of FAODs obtained by replacing amino acids in the amino acid sequence shown in SEQ ID NO:1 (hereinafter referred to as “Examples”), and FAODs containing the amino acid sequence shown in SEQ ID NO:1 (hereinafter referred to as "wild type") were evaluated.
• 50 ⁇ L of HEPES buffer solution containing 0.01 to 0.1 mg/mL of each enzyme (hereinafter referred to as "enzyme dilution solution”) was dispensed into tubes and kept on ice or on a heat block at 45°C for 15 minutes.
• the activity improvement rate and thermal stability improvement rate of the enzyme of the example before and after heat treatment are shown below. Compared to the wild-type enzyme, the enzyme of the example was confirmed to have at least one of the following effects: improved activity before heat treatment, improved activity after heat treatment, and improved thermal stability.
• an example with multiple amino acid substitutions was prepared, and its enzyme activity and thermal stability were evaluated.
• the example prepared is a mutant with two amino acid substitutions, N5L and G225N, in the amino acid sequence shown in SEQ ID NO:1.
• the evaluation method is as described above. As shown below, a significant improvement in activity and thermal stability after heat treatment was confirmed, compared to mutants with a single amino acid substitution, N5L or G225N.
• the activity and thermal stability were evaluated when not immobilized on a support.
• the evaluation method was as described above, and heat treatment was performed at 45°C.
• the activity improvement rate of the enzyme of the example after heat treatment is shown below. It was confirmed that the enzyme of the example had improved activity after heat treatment compared to the wild-type enzyme.
• a FAOD variant was produced to confirm whether activity and thermal stability were improved.
• a FAOD containing the amino acid sequence shown in SEQ ID NO: 199 was used as the other FAOD.
• This FAOD is a fructosyl amino acid oxidase derived from Aspergillus fumigatus [Accession Number: AAB88209], and is also called amadoriase I.
• the amino acid sequence of this FAOD was found to have 79.2% amino acid homology with the amino acid sequence shown in SEQ ID NO: 1.
• the FAOD and its variants were prepared using the same method as described above, and the activity improvement rate before heat treatment, the activity improvement rate after heat treatment, and the thermal stability improvement rate were evaluated. As shown in Table 17, it was confirmed that the variant obtained by replacing methionine at position 58 with another amino acid had at least one of the following effects compared to the wild-type enzyme: improved activity before heat treatment, improved activity after heat treatment, and improved thermal stability.
• the activity improvement rate and thermal stability improvement rate of the enzyme of the example before and after heat treatment are shown below.
• the enzyme of the example immobilized on a support was confirmed to have at least one of the following effects: improved activity before heat treatment, improved activity after heat treatment, and improved thermal stability.
• the activity and thermal stability of another example in a state immobilized on a support were evaluated.
• the evaluation method was as described above, and heat treatment was performed at 45°C.
• the activity improvement rate and thermal stability improvement rate of the enzyme of the example before and after heat treatment are shown below.
• the enzyme of the example in a state immobilized on a support was confirmed to have at least one of the following effects: improved activity before heat treatment, improved activity after heat treatment, and improved thermal stability.
• a comparative example (FAOD-E, Kikkoman Corporation) was used to evaluate activity and thermal stability when immobilized on a support.
• Bovine serum albumin (BSA) was used as the support, and activity was measured using the method described above.
• the heat treatment temperature was 45°C.
• the residual activity rate and thermal stability improvement rate after heat treatment were calculated using the following formulas.
• the table below shows the residual activity rates of the comparative examples and the wild type, and the residual activity rates and thermal stability improvement rates of the examples. In all examples, a significant improvement in thermal stability was confirmed compared to the wild type.
• the wild-type FAOD was a FAOD having the amino acid sequence shown in SEQ ID NO: 1
• the modified FAOD was a FAOD having an amino acid substitution of M58F in the wild-type FAOD, i.e., a FAOD having the amino acid sequence shown in SEQ ID NO: 38.
• a platinum electrode was subjected to silane coupling treatment to introduce amino groups onto the electrode surface.
• the above FAOD was added to a TES buffer solution in which bovine serum albumin (BSA) was dissolved, stirred and dissolved, and further impurities were removed by centrifugal filtration to obtain an enzyme solution.
• BSA bovine serum albumin
• a predetermined amount of glutaraldehyde solution was added to this enzyme solution and quickly stirred. 1 ⁇ L of this solution was dropped onto the platinum electrode with the amino group introduced, and thoroughly dried to obtain a sensor equipped with FAOD and BSA as a support.
• Four sensors were prepared for each of the wild-type FAOD and the modified FAOD, resulting in a total of eight sensors.
• the senor of this example is a sensor that includes a support and a FAOD variant fixed to the support, and after leaving the sensor undisturbed for one day under conditions of 75°C and 60% relative humidity, an output value of 73% was maintained, after leaving the sensor undisturbed for two days under the same conditions, an output value of 61% was maintained, after leaving the sensor undisturbed for three days under the same conditions, an output value of 49% was maintained, and after leaving the sensor undisturbed for 6 to 7 days under the same conditions, an output value of 27% was maintained.

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