Fructosyl-amino acid oxidase, gene, expression vector, glycated protein sensor, method for measuring glycated protein, and method for producing fructosyl-amino acid oxidase

Abstract

[Problem] In a glycated protein sensor provided with an enzyme immobilized thereon, a technology for enhancing the measurement accuracy and the stability of the glycated protein sensor is essential. However, no enzyme capable of enhancing the accuracy or stability of measurements using glycated protein sensors was known. [Solution] The present invention provides a fructosyl-amino acid oxidase that comprises an amino acid sequence having such a structure that, when aligned with the amino acid sequence represented by SEQ ID NO: 1, any amino acid residue corresponding to a position selected from the group consisting of position-35, position-71, position-101, position-117, position-203, position-216, position-217, position-247, and position-417 in the amino acid sequence represented by SEQ ID NO: 1 is substituted.

Description

Fructosyl amino acid oxidase, gene, expression vector, glycated protein sensor, method for measuring glycated protein, and method for producing fructosyl amino acid oxidase

[0001] 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 equipped with fructosyl amino acid oxidase immobilized on a support, a method for measuring glycated protein for detecting glycated protein by the reaction of fructosyl amino acid oxidase immobilized on a support, and a method for producing fructosyl amino acid oxidase.

[0002] The measurement of glycated protein is carried out as a diagnostic index for diabetes and a management index for blood glucose control. As an example, glycated hemoglobin and glycated albumin are frequently measured in clinical practice. As methods for measuring glycated protein, electrophoresis, high-performance liquid chromatography, immunoassay, enzyme method, etc. are known.

[0003] The method for measuring glycated protein by the enzyme method is as follows: in the first step, the protein is decomposed into amino acids or peptides by protease, and in the second step, fructosyl amino acid oxidase is allowed to act on the glycated amino acids (hereinafter sometimes also referred to as "glycated amino acids" or "fructosyl amino acids") or peptides containing glycated amino acids (hereinafter sometimes also referred to as "glycated peptides" or "fructosyl peptides") among those amino acids or peptides to generate hydrogen peroxide, and in the third step, the hydrogen peroxide is converted into a color reaction to measure the absorbance, or electrons released by the decomposition of hydrogen peroxide at an electrode are detected (see Patent Document 1).

[0004] Many inventions are known that feature enzymes modified to improve the measurement accuracy of specific glycated amino acids and glycated peptides. Examples include: an amadriase that can accurately measure glycated hemoglobin in samples containing glycated abnormal hemoglobin, corresponding to various genotypes (see Patent Document 2); an amadriase obtained by substituting amino acids in a specific amino acid sequence to improve specific activity with respect to glycation substrates (see Patent Document 3); an amadriase obtained by substituting amino acids in a specific amino acid sequence to retain activity in the presence of a surfactant (see Patent Documents 4 and 5); a fructosyl amino acid oxidase obtained by substituting amino acids in a specific amino acid sequence to have high thermal stability (see Patent Documents 6 and 7); an amadriase that is less affected by oxygen concentration (see Patent Document 8); a method for measuring fructosyllysine using fructosyl amino acid oxidase possessed by Aspergillus oryzae RIB40 strain (see Patent Document 9); and a fructosyl amino acid oxidase with high thermal stability when immobilized on a support (see Patent Document 10).

[0005] International Publication No. 2019 / 221264, International Publication No. 2019 / 045052, International Publication No. 2016 / 159384, International Publication No. 2016 / 072520, International Publication No. 2015 / 020200, International Publication No. 2015 / 096621, Japanese Patent Publication No. 2015-177790, International Publication No. 2016 / 063984, Japanese Patent Publication No. 2009-000084, International Publication No. 2023 / 145689

[0006] In enzyme-equipped glycated protein sensors, technologies to improve measurement accuracy and sensor stability are essential. Therefore, the problem that this invention aims to solve is to provide a fructosyl amino acid oxidase that possesses physicochemical properties such as high enzyme activity and high thermal stability that contribute to the accuracy of measurements and the stability of sensors using glycated protein sensors.

[0007] The inventors of this invention discovered a fructosyl amino acid oxidase with novel physicochemical properties from among genes with unknown functions. Furthermore, they found that substituting specific amino acids on known fructosyl amino acid oxidases improved their physicochemical properties, leading to the present invention.

[0008] In other words, the present invention provides a fructosyl amino acid oxidase having an amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence satisfies one or more of the following (1) to (10): (1) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 35 of SEQ ID NO: 1 is aspartic acid (D). (2) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 71 of SEQ ID NO: 1 is selected from arginine (R), lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (3) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 101 of SEQ ID NO: 1 is selected from phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), and isoleucine (I). (4) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 117 of SEQ ID NO: 1 is serine (S). (5) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 203 of SEQ ID NO: 1 is valine (V). (6) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 216 of SEQ ID NO: 1 is histidine (H). (7) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 217 of SEQ ID NO: 1 is arginine (R). (8) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 247 of SEQ ID NO: 1 is one of glutamic acid (E), arginine (R), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (9) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 417 of SEQ ID NO: 1 is serine (S). (10) When aligned with the amino acid sequence shown in Sequence ID No. 1, the amino acid corresponding to position 58 of Sequence ID No. 1 is phenylalanine (F).

[0009] One embodiment of the fructosyl amino acid oxidase of the present invention is the fructosyl amino acid oxidase described above, wherein it has an amino acid sequence having 79% or more homology to the amino acid sequence shown in SEQ ID NO: 1, and another embodiment is a fructosyl amino acid oxidase having an amino acid sequence having 95% or more homology to the amino acid sequence shown in SEQ ID NO: 1.

[0010] One embodiment of the fructosyl amino acid oxidase of the present invention is the fructosyl amino acid oxidase described above, which has higher enzyme activity and / or thermal stability compared to the fructosyl amino acid oxidase containing the amino acid sequence shown in Sequence ID No. 1. The enzyme activity is the enzyme activity of the fructosyl amino acid oxidase, i.e., FAOD activity, and the thermal stability is stability to a temperature of 45°C.

[0011] One embodiment of the fructosyl amino acid oxidase of the present invention is a fructosyl amino acid oxidase that satisfies at least (2) and (3) above. In another embodiment, the fructosyl amino acid oxidase may also satisfy at least (2) and (3) above, wherein (2) is a fructosyl amino acid oxidase in which, when aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 71 of SEQ ID NO: 1 is selected from arginine (R), lysine (K), and histidine (H).

[0012] One embodiment of the fructosyl amino acid oxidase of the present invention is a fructosyl amino acid oxidase that satisfies at least (1), (2) and (3), a fructosyl amino acid oxidase that satisfies at least (2), (3) and (10), or a fructosyl amino acid oxidase that satisfies at least (1), (2), (3) and (10). Here, (2) may be any of arginine (R), lysine (K), and histidine (H) when aligned with the amino acid sequence shown in SEQ ID NO: 1.

[0013] Furthermore, one embodiment of the fructosyl amino acid oxidase of the present invention is a fructosyl amino acid oxidase that satisfies at least (2)(3)(6), (2)(3)(7), (2)(3)(6)(7), (1)(2)(3)(6)(7), (1)(2)(3)(6)(7), (2)(3)(6)(10), (2)(3)(7)(10), (2)(3)(6)(7)(10), (1)(2)(3)(6)(7)(10), (1)(2)(3)(7)(10), or (1)(2)(3)(6)(7)(10). Here, (2) above may be such that, when aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 71 of SEQ ID NO: 1 is selected from arginine (R), lysine (K), and histidine (H).

[0014] One embodiment of the present invention may be a fructosyl amino acid oxidase having the amino acid sequence shown in any of SEQ ID NOs: 2 to 278. Another embodiment of the present invention may be a fructosyl amino acid oxidase having an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, added and / or inserted in the amino acid sequence shown in any of SEQ ID NOs: 2 to 278, and which satisfies one or more of the above-mentioned (1) to (10).

[0015] Furthermore, the present invention provides a gene encoding the amino acid sequence of the fructosyl amino acid oxidase described above, and an expression vector containing the gene.

[0016] Another aspect of the present invention provides a glycated protein sensor comprising the fructosyl amino acid oxidase described above and a support on which the fructosyl amino acid oxidase is immobilized. In the glycated protein sensor, the fructosyl amino acid oxidase can be immobilized on the support by crosslinking with an amine-reactive crosslinking agent.

[0017] Another aspect of the present invention provides a method for measuring glycated proteins by detecting them through the reaction of fructosyl amino acid oxidase as described above.

[0018] Another aspect of the present invention provides a method for producing fructosyl amino acid oxidase, which includes an amino acid substitution step in which, when the amino acid sequence of fructosyl amino acid oxidase is aligned with the amino acid sequence shown in Sequence ID No. 1, an amino acid is substituted with an amino acid selected from the group consisting of positions 35, 58, 71, 101, 117, 203, 216, 217, 247, and 417 in the amino acid sequence shown in Sequence ID No. 1.

[0019] In one embodiment, the amino acid substitution step may be one or more of (1) to (10). (1) The amino acid corresponding to position 35 of SEQ ID NO: 1 when aligned with the amino acid sequence shown in SEQ ID NO: 1 is substituted with aspartic acid (D). (2) The amino acid corresponding to position 71 of SEQ ID NO: 1 when aligned with the amino acid sequence shown in SEQ ID NO: 1 is substituted with any of the following selected: arginine (R), lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (3) The amino acid corresponding to position 101 of SEQ ID NO: 1 when aligned with the amino acid sequence shown in SEQ ID NO: 1 is substituted with any of the following selected: phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), and isoleucine (I). (4) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 117 of SEQ ID NO: 1 is replaced with serine (S). (5) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 203 of SEQ ID NO: 1 is replaced with valine (V). (6) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 216 of SEQ ID NO: 1 is replaced with histidine (H). (7) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 217 of SEQ ID NO: 1 is replaced with arginine (R). (8) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 247 of SEQ ID NO: 1 is replaced with one of the following selected from glutamic acid (E), arginine (R), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (9) The amino acid corresponding to position 417 of SEQ ID NO: 1 when aligned with the amino acid sequence shown in SEQ ID NO: 1 is replaced with serine (S). (10) The amino acid corresponding to position 58 of SEQ ID NO: 1 when aligned with the amino acid sequence shown in SEQ ID NO: 1 is replaced with phenylalanine (F).

[0020] In one embodiment of the present invention, in the method for producing fructosyl amino acid oxidase, the amino acid sequence of fructosyl amino acid oxidase before or after amino acid substitution may be an amino acid sequence having 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more homology with the amino acid sequence shown in Sequence ID No. 1.

[0021] The fructosyl amino acid oxidase of the present invention is heat-stable both when immobilized on a support and / or when not immobilized on a support. In particular, the heat stability of the fructosyl amino acid oxidase of the present invention when immobilized on a support is advantageous in that it improves the long-term stability of a glycated protein sensor equipped with the immobilized enzyme. Since the decrease in enzyme activity during storage is suppressed, the glycated protein sensor can provide highly reliable measurement results over a long period of time.

[0022] Furthermore, the present invention provides a fructosyl amino acid oxidase with higher enzyme activity and thermal stability compared to wild-type fructosyl amino acid oxidase. Additionally, the production method of the present invention allows for the production of modified compounds with high enzyme activity and / or thermal stability from known fructosyl amino acid oxidases.

[0023] This is a schematic diagram of a glycated protein sensor 10, which is one embodiment of the present invention. The diagram shows the output values ​​(left) and the rate of change in output values ​​(right) for 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 (modified M58F) having the amino acid sequence shown in SEQ ID NO: 2. The statistical significance of the difference between the two groups was calculated using a t-test. In the diagram, "***" indicates that the p-value was less than 0.001, "**" indicates that the p-value was less than 0.01, and "ns" indicates that no statistically significant difference was confirmed.

[0024] The fructosyl amino acid oxidase of the present invention has high enzyme activity and / or high thermal stability. Furthermore, the fructosyl amino acid oxidase according to one embodiment of the present invention has high enzyme activity and / or high thermal stability 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. Furthermore, the present invention provides a glycated protein sensor comprising a support and fructosyl amino acid oxidase immobilized on the support, a method for measuring glycated proteins by the reaction of fructosyl amino acid oxidase immobilized on the support, and a method for producing fructosyl amino acid oxidase. The present invention will be described in detail below based on embodiments.

[0025] Fructosyl amino acid oxidase is an enzyme that acts on amino acids with α-amino groups and / or ε-amino groups that have been glycated, or on peptides containing such amino acids, and generates hydrogen peroxide in the process of deglycosylating the glycated amino acids. Fructosyl amino acid oxidase (sometimes referred to as "FAOD") is also called amadriase, ketoamine oxidase, or fructosylamine oxidase. Fructosyl amino acid oxidase includes fructosyl peptide oxidase (sometimes referred to as "FPOD" or "FPOX"), which acts on glycated peptides.

[0026] Fructosyl amino acid oxidases use glycated amino acids and / or glycated peptides as substrates. Examples of glycated amino acids include ε-fructosyllysine (sometimes referred to as "fructosyllysine"), α-fructosylvaline (sometimes referred to as "fructosylvaline"), α-fructosylglycine (sometimes referred to as "fructosylglycine"), α-fructosylhistidine (sometimes referred to as "fructosylhistidine"), α-fructosylleucine (sometimes referred to as "fructosylleucine"), and α-fructosylserine (sometimes referred to as "fructosylserine").

[0027] Examples of glycated peptides include peptides consisting of 2 to 10 amino acids, preferably 2 to 6 amino acids, more preferably 2 to 3 amino acids, and containing one or more glycated amino acids. For example, α-fructosylvalylhistidine (sometimes referred to as "fructosylvalylhistidine") is one such example.

[0028] 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 effective pH range of pH 5 to 9, an effective temperature range of 20 to 80°C, and is soluble in buffer solutions. Specific examples of buffer solutions include Tris hydrochloride buffer and phosphate-buffered saline (PBS).

[0029] The fructosyl amino acid oxidase of the present invention may react specifically with a particular glycated amino acid or a peptide containing such amino acid. In one embodiment, the fructosyl amino acid oxidase has high specificity for fructosyllysine. High specificity for fructosyllysine means that when the reactivity to fructosyllysine or a peptide containing fructosyllysine is set to 100, the reactivity to other glycated amino acids or peptides containing other glycated amino acids is 20 or less. Here, 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. Other glycated amino acids refer to, for example, one or more selected from fructosylvaline, fructosylglycine, fructosylhistidine, fructosylleucine, and fructosylserine.

[0030] Fructosyl amino acid oxidases are classified into three groups based on their substrate specificity. Fructosyl amino acid oxidases belonging to Group 1 have high specificity for amino acids with a glycated α-amino group and / or peptides containing such amino acids. Fructosyl amino acid oxidases belonging to Group 2 have high specificity for amino acids with a glycated ε-amino group and / or peptides containing such amino acids. Fructosyl amino acid oxidases belonging to Group 3 have high specificity for amino acids with a glycated α-amino group and / or peptides containing such amino acids, as well as for amino acids with a glycated ε-amino group and / or peptides containing such amino acids. The fructosyl amino acid oxidase of the present invention may belong to any of these groups.

[0031] In another embodiment, the fructosyl amino acid oxidase exhibits high specificity for fructosylvalylhistidine. High specificity for fructosylvalylhistidine means that when the reactivity to fructosylvalylhistidine is set to 100, the reactivity to other glycated amino acids or other glycated peptides is 20 or less. Here, 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. Other glycated amino acids refer to, for example, one or more selected from fructosylvaline, fructosyllysine, fructosylglycine, fructosylhistidine, fructosylleucine, and fructosylserine, and other fructosyl peptides refer to peptides containing glycated amino acids other than fructosylvalylhistidine.

[0032] The fructosyl amino acid oxidase of the present invention may react specifically with a particular glycated amino acid or a peptide containing such amino acid. In one embodiment, the fructosyl amino acid oxidase has high specificity for fructosylvaline. High specificity for fructosylvaline means that when the reactivity to fructosylvaline or a peptide containing fructosylvaline is set to 100, the reactivity to other glycated amino acids or peptides containing other glycated amino acids is 20 or less. Here, 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. Other glycated amino acids refer to, for example, one or more selected from fructosyllysine, fructosylglycine, fructosylhistidine, fructosylleucine, and fructosylserine.

[0033] The amino acid sequence shown in Sequence ID No. 1 is the amino acid sequence of a fructosyl amino acid oxidase derived from Aspergillus pseudotamarii of the genus Aspergillus. Previously, Sequence ID No. 1 was expected to be the amino acid sequence of a type of FAD-dependent oxidoreductase based on sequence homology [Accession Number: XP_031920077], but it was not known until the present inventors revealed that a protein containing the amino acid sequence shown in Sequence 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 Sequence ID No. 1 has high thermal stability when immobilized on a support and high substrate specificity for specific glycated amino acids and / or glycated peptides (see Patent Document 10).

[0034] The fructosyl amino acid oxidase of the present invention is an enzyme having an amino acid sequence with high homology to the amino acid sequence shown in SEQ ID NO: 1. An amino acid sequence with high homology to SEQ ID NO: 1 is defined as having 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, and 71% or less of the amino acid sequence of SEQ ID NO: 1. The above may include amino acid sequences exhibiting homology of 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, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. These homology values ​​are rounded to one decimal place (the same applies below). Amino acid sequence homology can be calculated, for example, using the default parameters in BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) at the National Center for Biotechnology Information (NCBI).

[0035] One embodiment of fructosyl amino acid oxidase has an amino acid sequence with high homology to SEQ ID NO: 1 and contains or comprises an amino acid sequence that satisfies one or more of the following (1) to (10): (1) Position 35 is aspartic acid (D). (2) The amino acid at position 71 is selected from arginine (R), lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (3) The amino acid at position 101 is selected from phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), and isoleucine (I). (4) The amino acid at position 117 is serine (S). (5) The amino acid at position 203 is valine (V). (6) The amino acid at position 216 is histidine (H). (7) The amino acid at position 217 is arginine (R). (8) The amino acid at position 247 is one of the following: glutamic acid (E), arginine (R), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (9) The amino acid at position 417 is serine (S). (10) The amino acid at position 58 is phenylalanine (F).

[0036] On the other hand, fructosyl amino acid oxidases known prior to the priority date of this application are excluded from the fructosyl amino acid oxidases of the present invention, even if they satisfy any of the above conditions (1) to (10).

[0037] Here, the positions of the amino acids mentioned above are relative to the amino acid sequence shown in Sequence ID No. 1. In this specification, unless otherwise specified, the positions of amino acids are indicated relative to the amino acid sequence shown in Sequence ID No. 1. The positions of amino acid sequences other than Sequence ID No. 1 can be determined by aligning those amino acid sequences with the amino acid sequence shown in Sequence ID No. 1.

[0038] The fructosyl amino acid oxidase of another embodiment has an amino acid sequence highly homologous to SEQ ID NO: 1 and is a fructosyl amino acid oxidase satisfying a plurality of the above (1) to (10). As shown in the examples described later, it was confirmed that the fructosyl amino acid oxidase containing an amino acid sequence satisfying (2) and (3) has extremely high thermal stability as compared with the fructosyl amino acid oxidase containing the amino acid sequence shown in SEQ ID NO: 1. Furthermore, by introducing an amino acid substitution satisfying one or more selected from (1), (5), (6), (7) and (10) into the fructosyl amino acid oxidase containing an amino acid sequence satisfying (2) and (3), the thermal stability of the enzyme was further improved.

[0039] Among the fructosyl amino acid oxidases in the embodiments of the present invention, some specific examples of the amino acid sequences are shown. The amino acid sequences shown below are obtained by introducing one or more amino acid substitutions into the amino acid sequence shown in SEQ ID NO: 1.

[0040]

[0041] The amino acid sequences shown below are the amino acid sequence obtained by introducing the amino acid substitutions of W71R and A101F into the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequences obtained by further introducing one or more amino acid substitutions into the amino acid sequence. In the amino acid sequences shown below, the amino acid at position 71 is arginine (R), but the amino acid may be any one selected from lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A) and phenylalanine (F), and may be lysine (K) or histidine (H).

[0042]

[0043] The amino acid sequences shown below are amino acid sequences obtained by introducing an amino acid substitution of E216H into SEQ ID NO: 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and substituting the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0044]

[0045] The amino acid sequences shown below are amino acid sequences obtained by introducing amino acid substitutions of N35D and E216H into SEQ ID NO: 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and substituting the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0046]

[0047] The amino acid sequences shown below are amino acid sequences obtained by introducing an amino acid substitution of H217R into SEQ ID NO: 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and substituting the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0048]

[0049] The amino acid sequence shown below is obtained by introducing the amino acid substitutions N35D and H217R into Sequence ID No. 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0050]

[0051] The amino acid sequence shown below is obtained by introducing the amino acid substitutions E216H and H217R into Sequence ID No. 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0052]

[0053] The amino acid sequence shown below is obtained by introducing the amino acid substitutions N35D, E216H, and H217R into Sequence ID No. 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0054]

[0055] The amino acid sequence shown below is obtained by introducing the amino acid substitutions M58F and E216H into Sequence ID No. 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0056]

[0057] The amino acid sequence shown below is obtained by introducing the amino acid substitutions N35D and E216H into Sequence ID No. 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0058]

[0059] The amino acid sequence shown below is obtained by introducing the amino acid substitutions M58F and H217R into Sequence ID No. 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0060]

[0061] The amino acid sequence shown below is obtained by introducing the amino acid substitutions N35D, M58F, and H217R into Sequence ID No. 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0062]

[0063] The amino acid sequence shown below is obtained by introducing the amino acid substitutions M58F, E216H, and H217R into Sequence ID No. 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0064]

[0065] The amino acid sequence shown below is obtained by introducing the amino acid substitutions N35D, M58F, E216H and H217R into Sequence ID No. 1, and further substituting the tryptophan (W) at position 71 with arginine (R), lysine (K), or histidine (H), and the alanine (A) at position 101 with phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), or isoleucine (I).

[0066]

[0067] Another embodiment of the present invention is a fructosyl amino acid oxidase having an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted in the amino acid sequence shown in any of the above sequence numbers, and which has physicochemical properties such as high thermal stability and high substrate specificity for specific glycated amino acids and glycated peptides. Here, one or more amino acids means 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 amino acids.

[0068] The amino acid sequence of another embodiment of fructosyl amino acid oxidase may be an amino acid sequence in which one or more amino acids are modified or mutated, or deleted, substituted, added and / or inserted in the amino acid sequence shown in any of the above sequence numbers, and which satisfies the above conditions (1) to (10).

[0069] Furthermore, the present invention provides a gene comprising a nucleotide sequence encoding the above-described amino acid sequence. Here, the gene may be DNA or RNA, and may be complementary DNA (cDNA). Furthermore, the present invention provides an expression vector comprising a gene comprising a nucleotide sequence encoding the following amino acid sequence.

[0070] The fructosyl amino acid oxidase of the present invention may be a recombinant protein obtained by introducing a nucleic acid containing the base sequence encoding the fructosyl amino acid oxidase of the present invention into a host such as E. coli, yeast, mammalian cells, or insect cells and expressing it. Furthermore, the recombinant protein may also be a recombinant protein synthesized in a cell-free protein synthesis system.

[0071] In one embodiment of the present invention, the enzyme is a fructosyl amino acid oxidase containing the above-described amino acid sequence. In this embodiment, an amino acid sequence that functions as a tag may be added to the N-terminal and / or C-terminal side to facilitate the purification and detection of the enzyme. Examples of tags include His tag, Myc tag, HA tag, FLAG tag, etc.

[0072] A fructosyl amino acid oxidase according to one embodiment of the present invention exhibits high enzymatic activity toward a substrate when immobilized on a support and / or when not immobilized on a support. In one embodiment of the present invention, the fructosyl amino acid oxidase obtained by the above-described amino acid substitution includes those that exhibit higher enzymatic activity toward a substrate than fructosyl amino acid oxidase consisting of an amino acid sequence without amino acid substitution. The fructosyl amino acid oxidase of the present invention may have enzymatic activity that is 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more higher than the fructosyl amino acid oxidase before amino acid substitution (i.e., wild type). These values ​​are rounded to one decimal place.

[0073] Furthermore, the rate of activity improvement of the fructosyl amino acid oxidase of the present invention compared to the wild type can be calculated, for example, using the following formula. The calculated rate of activity improvement will be a positive value if the activity is improved by the amino acid substitution, and a negative value if the activity is decreased by the amino acid substitution.

[0074]

[0075] Another embodiment of fructosyl amino acid oxidase is a fructosyl amino acid oxidase in which the enzyme activity measured after heat treatment, both when immobilized on a support and / or when not immobilized on a support, is higher than that of the wild type. The fructosyl amino acid oxidase of this embodiment may have an improved enzyme activity after heat treatment of 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 (i.e., the wild type). These values ​​are rounded to one decimal place (the same applies hereinafter).

[0076] In one embodiment, the fructosyl amino acid oxidase is thermally stable when immobilized on a support and / or when not immobilized on a support. In another embodiment, the fructosyl amino acid oxidase obtained by the above-described amino acid substitution is more thermally stable than the fructosyl amino acid oxidase consisting of an amino acid sequence without amino acid substitution. Specifically, the fructosyl amino acid oxidase of the present invention may have improved thermal stability by 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more compared to the fructosyl amino acid oxidase before amino acid substitution (i.e., wild type). These values ​​are rounded to one decimal place (the same applies hereinafter).

[0077] The thermal stability of the fructosyl amino acid oxidase of the present invention, compared to the wild type, can be calculated, for example, using the following formula. The following formula compares the residual activity of two FAODs after heat treatment. The residual activity may be the value obtained by dividing the enzyme activity value after heat treatment by the enzyme activity value before heat treatment. The calculated thermal stability improvement rate will be a positive value if thermal stability is improved by amino acid substitution, and a negative value if thermal stability is decreased by amino acid substitution.

[0078]

[0079] One of the characteristics of fructosyl amino acid oxidases containing the amino acid sequence shown in Sequence ID No. 1 is that they are 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 while immobilized on a support, and is characterized by having a residual activity of 70% or more after treatment at 65°C.

[0080] In one embodiment of the present invention, a fructosyl amino acid oxidase containing an amino acid sequence obtained by substituting one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 with another amino acid may include one that has higher enzyme activity when immobilized on a support and / or when not immobilized on a support than a fructosyl amino acid oxidase containing the amino acid sequence shown in SEQ ID NO: 1.

[0081] In another embodiment of the present invention, a fructosyl amino acid oxidase containing an amino acid sequence obtained by substituting one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 with another amino acid may have higher enzyme activity after heat treatment when immobilized on a support and / or when not immobilized on a support than the fructosyl amino acid oxidase containing the amino acid sequence shown in SEQ ID NO: 1. Here, heat treatment may refer to conditions in which the enzyme is heated to 45°C to 48°C for 15 minutes.

[0082] In another embodiment of the present invention, a fructosyl amino acid oxidase containing an amino acid sequence obtained by substituting one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 with other amino acids may have higher thermal stability when immobilized on a support and / or when not immobilized on a support than the fructosyl amino acid oxidase containing the amino acid sequence shown in SEQ ID NO: 1. Here, thermal stability is expressed as the residual activity when the enzyme is kept at 45°C to 48°C for 15 minutes.

[0083] Fructosyl amino acid oxidases containing the amino acid sequence shown in Sequence ID No. 1 exhibit significantly higher thermal stability when immobilized on a support compared to other FAODs. Therefore, even if the improvement in thermal stability during support immobilization due to the aforementioned amino acid substitution is only a few percent, the effect is considered remarkable.

[0084] 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 comprises at least the fructosyl amino acid oxidase described above. 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.

[0085] One embodiment of the present invention, fructosyl amino acid oxidase, can be included in a glycated protein sensor. An example of a glycated protein sensor comprises fructosyl amino acid oxidase and a support on which the fructosyl amino acid oxidase is immobilized. The fructosyl amino acid oxidase included in this glycated protein sensor has extremely high residual activity even after heat treatment of the protein sensor, so that its output value is maintained at a high temperature even when the sensor is placed in a high-temperature environment. Specifically, in the sensor of this embodiment, the output value of the sensor after being left at a temperature of 65°C or 80°C for 20 minutes may be 60% or more of the output value of the sensor before leaving it. Furthermore, the output value of the sensor after being left at a temperature of 75°C and relative humidity of 60% for one, two, or three days may be 40%, 50%, 60%, or 70% or more of the output value of the sensor before leaving it.

[0086] Examples of methods for immobilizing enzymes such as FAOD contained in the glycated protein sensor onto a support include covalent bonding, physical adsorption, ionic bonding, crosslinking, inclusion, and biochemical specific bonding. Depending on the enzyme used, an immobilization method that does not deactivate the enzyme may be selected, and multiple immobilization methods may be used in combination.

[0087] In some embodiments, FAOD is immobilized on a support by crosslinking with a crosslinking agent, either alone or in combination with a carrier. In such cases, the support is a protein other than FAOD, and the FAOD and support are mixed together before the FAOD is immobilized on the support using a crosslinking agent such as glutaraldehyde or an isocyanate derivative. Specific examples of proteins include albumin such as bovine serum albumin (BSA), collagen, and gelatin.

[0088] Crosslinking agents include substances that crosslink molecules intermolecularly or intramolecularly, specifically glutaraldehyde, isocyanate derivatives, formaldehyde, glyoxal, malondialdehyde, succinaldehyde, and the like. Among these, amine-reactive crosslinking agents are preferably used. Amine-reactive crosslinking agents are crosslinking agents that react with the amino groups of proteins to crosslink the proteins, specifically glutaraldehyde, formaldehyde, N-hydroxyesters, amide esters, imide esters, and the like.

[0089] In one embodiment, the glycation sensor comprises a FAOD and a support on which the FAOD is immobilized by an amine-reactive crosslinking agent. The amine-reactive crosslinking agent reacts with and crosslinks amino groups present on the surface of the fructosyl amino acid oxidase used in the glycation sensor.

[0090] In other embodiments, the support may be a synthetic polymer, resin, inorganic material, polysaccharide, mineral, clay, etc. Specifically, means for fixing FAOD to the support include fixing FAOD to a support if a fluororesin, ion exchange resin, polyvinyl alcohol resin, hydraulic resin, photocurable resin, solid polymer electrolyte, polyion complex, urethane, etc. is used as the support; fixing FAOD to a support if a charcoal, bone char, silica gel, glass, zeolite, Celite, alumina, titanium dioxide, ceramic, hydroxyapatite, etc. is used as the support by physical interaction between the support and the FAOD; fixing FAOD to a support by enclosing it in a semipermeable membrane such as nylon, cellophane, ethylcellulose, acetylcellulose, polystyrene, or a phospholipid membrane if a support is used; or fixing FAOD to a gel if a polyacrylamide gel, agar, gelatin, carrageenan, sodium alginate gel, calcium alginate gel, chitosan gel, etc. is used as the support.

[0091] Furthermore, the support material may be a carbon material and / or beads. The carbon material may be carbon nanotubes, fullerenes, graphene, etc. The beads may be carbon nanoparticles (carbon beads), silica (SiO2).２ The beads may consist of fine particles (silica beads), chitin, chitosan, or polysaccharides such as alginic acid. The beads may also contain metal particles or magnetizing materials, and may be magnetic beads. The average particle size of the beads may be 10 nm or more, or 200 nm or less. FAOD can be fixed to the beads by crosslinking.

[0092] A preferred embodiment of the glycated protein sensor of the present invention comprises a support, a fructosyl amino acid oxidase immobilized on the support, and a detection unit. The detection unit detects hydrogen peroxide produced from glycated amino acids and / or glycated peptides by the fructosyl amino acid oxidase. The glycated protein sensor can calculate the amount (concentration) of glycated protein from the detection result of the detection unit.

[0093] In one embodiment of the glycated protein sensor of the present invention, the detection unit is a hydrogen peroxide detection unit that detects hydrogen peroxide, and specifically, the detection unit may be a hydrogen peroxide electrode. Since the electrons released when hydrogen peroxide is decomposed into oxygen are detected as an electric current in the hydrogen peroxide electrode, the amount (concentration) of hydrogen peroxide can be calculated from the detected current value.

[0094] Figure 1 shows the configuration of a glycated protein sensor 10, which is one embodiment of the present invention. The glycated protein sensor 10 shown in Figure 1 has an enzyme layer 12 containing fructosyl amino acid oxidase and protease, and a hydrogen peroxide detection unit 14. The fructosyl amino acid oxidase and protease are crosslinked to bovine serum albumin, which is a support, by glutaraldehyde, which is a crosslinking agent. The bovine serum albumin is also crosslinked to each other by glutaraldehyde. These form the enzyme layer 12. The enzyme layer 12 is laminated to the detection surface 16 side of the hydrogen peroxide detection unit 14 and is bonded to the detection surface 16 of the hydrogen peroxide detection unit 14 by a silane coupling agent 18.

[0095] In another embodiment, the detection unit is an optical detection unit that detects hydrogen peroxide by measuring absorbance or light intensity. For example, hydrogen peroxide can be quantified by detecting a color reaction in the presence of peroxidase and an oxidative coloring dye, or by detecting the luminescence intensity with luminol.

[0096] In another embodiment, the detection unit is an electrochemiluminescence detection unit, and gold electrodes, platinum electrodes, or transparent electrodes made of indium tin oxide (ITO electrodes) are used. Hydrogen peroxide is detected by measuring the emission of light from a luminescent reagent such as luminol. The detection unit may be a hydrogen peroxide detection unit of any other type.

[0097] 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 non-body fluid solution, 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. For example, the solution may be a standard solution or a calibration solution. The solution may contain a buffer.

[0098] Body fluids may be blood, serum, plasma, lymph, interstitial fluids such as interstitial fluid, intercellular fluid, or interstitial fluid, or body cavity fluids, serosal fluids, pleural fluid, ascites, pericardial fluid, cerebrospinal fluid, synovial fluid, or aqueous humor. Body fluids may be digestive fluids such as saliva, gastric juice, bile, pancreatic juice, or intestinal juice, or sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, or milk. Body fluids may be animal body fluids or human body fluids. Body fluids may be liquids in foods containing animal-derived proteins (e.g., milk or dairy products). Body fluids may be plant sap, plant biosap, or plant-derived liquids. For example, body fluids may be plant fruit juice, nectar, or sap.

[0099] The solution may contain the substance to be measured. For example, the solution may be tears, and the substance to be measured may be albumin or glycoalbumin contained in tears. Alternatively, the substance to be measured may be albumin, glycoalbumin, hemoglobin, or glycated hemoglobin in blood, serum, or plasma; albumin or glycoalbumin in interstitial fluid; albumin or glycoalbumin in urine; or albumin or glycoalbumin in saliva.

[0100] The glycated protein sensor of the present invention measures glycated proteins. Glycated proteins may be fructosamines. Fructosamines are a general term for glycated proteins contained in blood, and specifically include glycated albumin and glycated hemoglobin contained in blood.

[0101] One embodiment of the present invention may include not only fructosyl amino acid oxidase immobilized on a support, but also protease immobilized on a support. Proteases are a general term for peptide bond hydrolases that hydrolyze and catabolize proteins and polypeptides. A protease may also be an enzyme that breaks down proteins into peptide fragments. When a protein contains glycated amino acid residues, the action of a protease may produce one or more selected from the group consisting of glycated amino acids, peptide fragments containing glycated amino acids, unglycated amino acids, and peptide fragments that do not contain glycated amino acids. Fructosyl amino acid oxidase may react with glycated amino acids or peptide fragments containing glycated amino acids to produce hydrogen peroxide.

[0102] In one embodiment of the present invention, a support on which an enzyme is immobilized forms a support layer, such as a thin film layer, and is stacked and positioned relative to 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 unit, thereby bonding the support and the detection surface.

[0103] Furthermore, the present invention provides a method for measuring glycated proteins. The method for measuring glycated proteins of the present invention is a method for measuring glycated proteins by detecting glycated proteins through the reaction of fructosyl amino acid oxidase immobilized on a support. The measurement method of the present invention may be a non-diagnostic and / or diagnostic measurement method.

[0104] Furthermore, the present invention 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, in which, when the amino acid sequence of fructosyl amino acid oxidase is aligned with the amino acid sequence shown in Sequence ID No. 1, one of the amino acids corresponding to a position selected from the group consisting of positions 35, 71, 101, 117, 203, 216, 217, 247, and 417 in the amino acid sequence shown in Sequence ID No. 1 is substituted with the amino acid sequence of fructosyl amino acid oxidase. The activity and / or thermal stability of the fructosyl amino acid oxidase produced by this method, both when immobilized on a support and / or when not immobilized on a support, is higher than that of fructosyl amino acid oxidase in which no amino acids have been substituted.

[0105] Here, the amino acid sequence of the fructosyl amino acid oxidase used has high homology to the amino acid sequence shown in SEQ ID NO: 1. That is, in one embodiment of the manufacturing method of the present invention, the amino acid sequence before and / or after amino acid substitution is an amino acid sequence that has high homology to the amino acid sequence shown in SEQ ID NO: 1. Specifically, an amino acid sequence with high homology to SEQ ID NO: 1 is one in which the amino acid sequence of SEQ ID NO: 1 has 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, and 7 The amino acid sequences may contain homology of 1% 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, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. These homology values ​​are rounded to one decimal place (the same applies below). The homology of amino acid sequences can be calculated, for example, using the default parameters in BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) at the National Center for Biotechnology Information (NCBI).

[0106] The amino acid substitution step in one embodiment of the manufacturing method of the present invention includes one or more selected from (1) to (10) below: (1) Substituting the amino acid corresponding to position 35 of SEQ ID NO: 1 when aligned with the amino acid sequence shown in SEQ ID NO: 1 with aspartic acid (D). (2) Substituting the amino acid corresponding to position 71 of SEQ ID NO: 1 when aligned with the amino acid sequence shown in SEQ ID NO: 1 with any of the following selected from arginine (R), lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (3) Substituting the amino acid corresponding to position 101 of SEQ ID NO: 1 when aligned with the amino acid sequence shown in SEQ ID NO: 1 with any of the following selected from phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), and isoleucine (I). (4) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 117 of SEQ ID NO: 1 is replaced with serine (S). (5) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 203 of SEQ ID NO: 1 is replaced with valine (V). (6) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 216 of SEQ ID NO: 1 is replaced with histidine (H). (7) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 217 of SEQ ID NO: 1 is replaced with arginine (R). (8) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 247 of SEQ ID NO: 1 is replaced with one of the following selected from glutamic acid (E), arginine (R), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (9) The amino acid corresponding to position 417 of SEQ ID NO: 1 when aligned with the amino acid sequence shown in SEQ ID NO: 1 is replaced with serine (S). (10) The amino acid corresponding to position 58 of SEQ ID NO: 1 when aligned with the amino acid sequence shown in SEQ ID NO: 1 is replaced with phenylalanine (F).

[0107] The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0108] 1. Analysis of the activity and thermal stability of fructosyl amino acid oxidase in examples not immobilized on a support The detailed production method of the fructosyl amino acid oxidase used in this example is described below. First, a gene was introduced into an expression vector having a lactose operon, containing a nucleotide sequence encoding one of the amino acid sequences described herein, and further designed to add a histidine tag to the N-terminus of each inserted protein. Escherichia coli BL21 (DE3) Derived strain (manufactured by Nippon Gene Co., Ltd.) was transformed using this expression vector.

[0109] Escherichia coli transformed in LB liquid medium containing 50 μg / mL kanamycin (Nacalai Tesque) was cultured with shaking for 2 hours. Furthermore, these Escherichia coli were cultured at 16°C for 20 hours in LB liquid medium supplemented with isopropyl β-D-thiogalactopyranoside (hereinafter referred to as "IPTG") to a final concentration of 0.1 mM. The collected Escherichia coli were suspended in buffer A (20 mM Tris-HCl (pH 7.5) containing 0.25 M sodium chloride and 20 mM imidazole), the cells were disrupted using an ultrasonic lysator, and the cell lysate supernatant was obtained by centrifugation. The supernatant of the cell disruption solution was filtered through a 0.45 μm pore size filter, then applied to EconoFit Nuvia IMAC Columns, Ni-charged (BIO-RAD), washed with 7 times the column volume 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).

[0110] The obtained eluate was dialyzed with 10 mM sodium phosphate buffer (pH 8.4) containing 150 mM sodium chloride and 50% by volume glycerol to obtain the sample. The protein concentration in the sample was measured using absorbance at 280 nm.

[0111] The activity and thermal stability of FAODs obtained by substituting any amino acid in the amino acid sequence shown in Sequence ID No. 1 (hereinafter referred to as "Examples") and FAODs containing the amino acid sequence shown in Sequence ID No. 1 (hereinafter referred to as "Wild Type") were evaluated. 50 μL of HEPES buffer containing 0.01 to 0.1 mg / mL of each enzyme (hereinafter referred to as "Enzyme Diluent") was dispensed into tubes and maintained at 45°C on ice or a heat block for 15 minutes. Next, to the wells of a microtiter plate containing 1 mM F-Lys, a 0.5 w / w% TOOS, POD / 4-AA solution (diluted with HEPES buffer (pH 8.0, 0.15 M NaCl) to 4.76 μg / mL peroxidase and 0.1 mg / mL 4-aminoantipyrine), and an enzyme dilution maintained on ice or heat-treated in a heat block were added at room temperature and mixed by pipetting. Using a microtiter plate reader set to 37°C, the absorbance at 555 nm was measured every 80 seconds for 20 minutes after addition, and the change in absorbance per unit time was calculated as the FAOD activity. Hereinafter, the activity measured using the enzyme maintained on ice will be referred to as "FAOD activity before heat treatment," and the activity measured using the enzyme after heat treatment in a heat block will be referred to as "FAOD activity after heat treatment."

[0112] Using the examples and the average value of wild-type FAOD activity, the rate of activity improvement before and after heat treatment, as well as the rate of thermal stability improvement, were calculated using the following formulas.

[0113]

[0114]

[0115]

[0116] The activity improvement rates and thermal stability improvement rates of the enzymes in the examples before and after heat treatment are shown below. Compared to the wild-type enzymes, the enzymes in Examples 1 to 10 showed improved activity before heat treatment, improved activity after heat treatment, and improved thermal stability. Furthermore, the enzyme in Example 11 showed improved activity and thermal stability after heat treatment, while the enzymes in Examples 12 and 13 showed improved activity before heat treatment.

[0117]

[0118] In addition to the enzymes disclosed in the table above, enzymes containing amino acid substitutions of K247E, K247Q, or K247A were also prepared, and it was confirmed that these enzymes were active.

[0119] Based on the results of the above examples, new enzymes with multiple amino acid substitutions were prepared, and their activity and thermal stability were evaluated. The improvement in activity and thermal stability of the enzymes in the examples, both before and after heat treatment, are shown below. Compared to the wild-type enzyme, the enzymes in the examples showed improved activity before and after heat treatment, as well as improved thermal stability.

[0120]

[0121] In particular, Examples 15-39, which included an amino acid substitution at position 71, showed a significantly higher improvement in activity after heat treatment compared to the wild type. Examples 3-7 clearly show that a significant improvement in activity after heat treatment can be observed by substituting the tryptophan (W) at position 71 of the wild type with highly polar amino acids such as arginine (R), lysine (K), histidine (H), glutamine (Q), or tyrosine (Y). Therefore, it was predicted that enzymes exhibiting equivalent or superior effects could be obtained even if the amino acid at position 71 in Examples 15-41 were glutamine (Q) or tyrosine (Y).

[0122] Furthermore, Examples 21 and 27 showed significantly higher activity before and after heat treatment compared to the wild type. Therefore, it was predicted that if the amino acid at position 71 of Examples 38 and 39 was substituted with lysine (K), or if the amino acid at position 101 was substituted with glutamic acid (E) or histidine (H), enzymes with activity equivalent to or greater than that of the enzymes in Example 38 or 39 could be obtained.

[0123] 2. Analysis of the activity and thermal stability of the fructosyl amino acid oxidase of the examples immobilized on a support The activity and thermal stability of the fructosyl amino acid oxidase of the examples and the wild type were evaluated in a state immobilized on a support. Bovine serum albumin (BSA) was used as the support. A Hepes buffer (10 mM Hepes + 150 mM NaCl, pH 8.0) containing 0.1 mg / mL of enzyme and 0.264% (w / v) BSA was prepared, and glutaraldehyde was added to make it 0.1% (w / v). After incubation at 25°C for 20 minutes, 90 μL of ice-cooled TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0-8.5) was added, and the reaction was terminated by adding an excess amount of Tris containing a primary amine. Enzyme activity was measured after maintaining on ice for 15 minutes or on a heat block at 45°C for 15 minutes. Using the average FAOD activity of each enzyme, the improvement in activity before and after heat treatment, as well as the improvement in thermal stability, were calculated.

[0124] The activity improvement rate and thermal stability improvement rate of the enzymes in the examples before and after heat treatment are shown below. Compared to the wild-type enzyme, the enzymes in the following examples, immobilized on a support, showed at least one of the following effects: improved activity before heat treatment, improved activity after heat treatment, and improved thermal stability.

[0125]

[0126]

[0127] In addition to the enzymes disclosed in the table above, enzymes containing amino acid substitutions of K247E, K247Q, or K247A were also prepared, and it was confirmed that these enzymes were active.

[0128] Examples 21 and 27 showed significantly higher activity before and after heat treatment during support fixation compared to the wild type. Therefore, it was predicted that if the amino acid at position 71 of Examples 38 and 39 was substituted with lysine (K), or if the amino acid at position 101 was substituted with glutamic acid (E) or histidine (H), enzymes with activity equivalent to or greater than that of the enzymes in Example 38 or 39 could be obtained.

[0129] The wild-type FAOD, consisting of the amino acid sequence shown in Sequence ID No. 1, exhibits significantly higher thermal stability when immobilized on a support compared to other FAODs. Therefore, even if the improvement in thermal stability in the examples is only slight, its effect is considered remarkable.

[0130] 4. Evaluation of Sensors Equipped with Wild-Type and Modified Fructosyl Amino Acid Oxidases A glycated protein sensor equipped with wild-type and modified FAODs was prepared, and its thermal stability was evaluated. For the wild-type FAOD, a FAOD having the amino acid sequence shown in SEQ ID NO: 1 was used. For the modified FAOD, a FAOD having the M58F amino acid substitution in the wild-type FAOD, i.e., a FAOD having the amino acid sequence shown in SEQ ID NO: 2, was used. A silane coupling treatment was performed on the platinum electrode to introduce amino groups to the electrode surface. The above FAOD was added to TES buffer containing dissolved bovine serum albumin (BSA), stirred and dissolved, and then contaminants were removed by centrifugal filtration to obtain an enzyme solution. A predetermined amount of glutaraldehyde solution was added to this enzyme solution and stirred quickly. 1 μL of this solution was dropped onto the platinum electrode with the introduced amino groups, and thoroughly dried to obtain a sensor equipped with FAOD and a support BSA. Four sensors were fabricated for both the wild-type FAOD and the modified FAOD, resulting in a total of eight sensors.

[0131] For each sensor, 200 μL of HEPES buffer containing 50 μM F-Lys was dropped, and the change in the resulting current value was recorded as the output value of the sensor. After washing each sensor with HEPES buffer, the moisture was removed with a blower, and the sensors were left to stand at a temperature of 75°C and a relative humidity of 60%. After a certain period, the sensors were removed and their output values ​​were recorded in the same manner. This standing and output value recording process was repeated for the same sensor on days 1, 2, 3, 6, and 7, and the output values ​​for each day were recorded. The evaluation results are shown in Figure 2. Compared to sensors with wild-type FAOD, sensors equipped with modified FAOD showed significantly higher output values ​​from day 1 to 7, and the rate of change with day 0 set as 100% was also significantly higher from day 1 to 7.

[0132] In other words, the sensor of this embodiment is a sensor comprising a support and a FAOD modified body fixed to the support. 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. After leaving the sensor undisturbed for six to seven days under the same conditions, an output value of 27% was maintained.

[0133] Furthermore, sensors containing the following FAOD modifiers were fabricated using the same method as described above, and the output values ​​from day 1 to day 7 were recorded using the same method under conditions of 75°C and 60% relative humidity. The obtained output values ​​were subjected to exponential regression, and the output value half-life relative to the output value on day 0 was calculated. The output value half-lives of sensors containing each modifier are shown in the table below. In the table, the relative output value half-life is the ratio of the half-life of the modifier to the output value half-life of the wild type, which is set to 1. Sensors containing FAOD modifiers showed an extension of 1.7 to 3.6 times the output value half-life compared to sensors containing the FAOD wild type. Therefore, it was shown that sensors with excellent heat resistance and durability can be obtained by using FAOD modifiers.

[0134]

[0135] While several embodiments and examples of the present disclosure have been described above, these embodiments and examples are illustrative in nature. For example, each of the above embodiments has been described in detail to make the present disclosure easier to understand, and dimensions, configurations, materials, and circuits may be added or modified as needed. Embodiments that arbitrarily combine one or more of the features of the present disclosure listed above are also included in the scope of the present disclosure. The claims encompass a number of variations on the embodiments without departing from the technical idea of ​​the present disclosure. Therefore, the embodiments and examples disclosed herein are provided for illustrative purposes only and should not be considered as limiting the scope of the present disclosure.

[0136] Furthermore, the present invention encompasses the following embodiments.

[0137] [Aspect 1-1] A fructosyl amino acid oxidase having an amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence satisfies the following conditions: - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 71 of SEQ ID NO: 1 is selected from arginine (R), lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 101 of SEQ ID NO: 1 is selected from phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), and isoleucine (I). [Aspect 1-2] The fructosyl amino acid oxidase according to Aspect 1-1, having an amino acid sequence with a homology of 79% or more to the amino acid sequence shown in SEQ ID NO: 1. [Aspect 1-3] A fructosyl amino acid oxidase according to Aspect 1-1, having an amino acid sequence that has 95% or more homology to the amino acid sequence shown in SEQ ID NO: 1. [Aspect 1-4] A gene encoding the amino acid sequence of a fructosyl amino acid oxidase according to any one of Aspects 1-1 to 1-3, or an expression vector containing the gene. [Aspect 1-5] A glycated protein sensor comprising a fructosyl amino acid oxidase according to any one of Aspects 1-1 to 1-3 and a support on which the fructosyl amino acid oxidase is immobilized. [Aspect 1-6] A method for measuring glycated proteins by a reaction of a fructosyl amino acid oxidase according to any one of Aspects 1-1 to 1-3.

[0138] [Aspect 2-1] A fructosyl amino acid oxidase having an amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence satisfies the following conditions: - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 58 of SEQ ID NO: 1 is phenylalanine (F). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 71 of SEQ ID NO: 1 is one of arginine (R), lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 101 of SEQ ID NO: 1 is one of phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), and isoleucine (I). [Aspect 2-2] A fructosyl amino acid oxidase according to Aspect 2-1, having an amino acid sequence having 79% or more homology to the amino acid sequence shown in SEQ ID NO: 1. [Aspect 2-3] A fructosyl amino acid oxidase according to Aspect 2-1, having an amino acid sequence having 95% or more homology to the amino acid sequence shown in SEQ ID NO: 1. [Aspect 2-4] A gene encoding the amino acid sequence of a fructosyl amino acid oxidase according to any one of Aspects 2-1 to 2-3, or an expression vector containing the gene. [Aspect 2-5] A glycated protein sensor comprising a fructosyl amino acid oxidase according to any one of Aspects 2-1 to 2-3 and a support on which the fructosyl amino acid oxidase is immobilized. [Aspect 2-6] A method for measuring glycated proteins by the reaction of a fructosyl amino acid oxidase according to any one of Aspects 2-1 to 2-3.

[0139] [Aspect 3-1] A fructosyl amino acid oxidase having an amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence satisfies the following conditions: - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 35 of SEQ ID NO: 1 is aspartic acid (D). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 71 of SEQ ID NO: 1 is one of arginine (R), lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 101 of SEQ ID NO: 1 is one of phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), and isoleucine (I). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 216 of SEQ ID NO: 1 is histidine (H). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 217 of SEQ ID NO: 1 is arginine (R). [Aspect 3-2] The fructosyl amino acid oxidase according to Aspect 3-1, having an amino acid sequence that has 79% or more homology to the amino acid sequence shown in SEQ ID NO: 1. [Aspect 3-3] The fructosyl amino acid oxidase according to Aspect 3-1, having an amino acid sequence that has 95% or more homology to the amino acid sequence shown in SEQ ID NO: 1. [Aspect 3-4] A gene encoding the amino acid sequence of the fructosyl amino acid oxidase according to any one of Aspects 3-1 to 3-3, or an expression vector containing the gene. [Aspect 3-5] A glycated protein sensor comprising the fructosyl amino acid oxidase according to any one of Aspects 3-1 to 3-3, and a support on which the fructosyl amino acid oxidase is immobilized. [Aspect 3-6] A method for measuring glycated proteins by the reaction of fructosyl amino acid oxidase as described in any of aspects 3-1 to 3-3.

[0140] [Aspect 4-1] A fructosyl amino acid oxidase having an amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence satisfies the following conditions: - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 35 of SEQ ID NO: 1 is aspartic acid (D). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 58 of SEQ ID NO: 1 is phenylalanine (F). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 71 of SEQ ID NO: 1 is one of arginine (R), lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 101 of SEQ ID NO: 1 is selected from phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), and isoleucine (I). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 216 of SEQ ID NO: 1 is histidine (H). - When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 217 of SEQ ID NO: 1 is arginine (R). [Aspect 4-2] The fructosyl amino acid oxidase according to Aspect 4-1, having an amino acid sequence that has 79% or more homology to the amino acid sequence shown in SEQ ID NO: 1. [Aspect 4-3] The fructosyl amino acid oxidase according to Aspect 4-1, having an amino acid sequence that has 95% or more homology to the amino acid sequence shown in SEQ ID NO: 1. [Aspect 4-4] A gene encoding the amino acid sequence of a fructosyl amino acid oxidase according to any one of aspects 4-1 to 4-3, or an expression vector containing the gene. [Aspect 4-5] A glycated protein sensor comprising a fructosyl amino acid oxidase according to any one of aspects 4-1 to 4-3 and a support on which the fructosyl amino acid oxidase is immobilized. [Aspect 4-6] A method for measuring glycated proteins by a reaction of a fructosyl amino acid oxidase according to any one of aspects 4-1 to 4-3.

[0141] 10. Glycated protein sensor 12. Enzyme layer 14. Hydrogen peroxide detection unit 16. Detection surface 18. Silane coupling agent

Claims

1. A fructosyl amino acid oxidase having an amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence satisfies one or more of the following (1) to (9): (1) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 35 of SEQ ID NO: 1 is aspartic acid (D). (2) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 71 of SEQ ID NO: 1 is selected from arginine (R), lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (3) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 101 of SEQ ID NO: 1 is selected from phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), and isoleucine (I). (4) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 117 of SEQ ID NO: 1 is serine (S). (5) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 203 of SEQ ID NO: 1 is valine (V). (6) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 216 of SEQ ID NO: 1 is histidine (H). (7) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 217 of SEQ ID NO: 1 is arginine (R). (8) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 247 of SEQ ID NO: 1 is one of glutamic acid (E), arginine (R), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (9) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 417 of SEQ ID NO: 1 is serine (S).

2. The fructosyl amino acid oxidase according to claim 1, having an amino acid sequence that has 79% or more homology to the amino acid sequence shown in Sequence ID No.

1.

3. The fructosyl amino acid oxidase according to claim 1, having an amino acid sequence that has 95% or more homology to the amino acid sequence shown in Sequence ID No.

1.

4. The fructosyl amino acid oxidase according to claim 3, which has higher enzyme activity compared to the fructosyl amino acid oxidase containing the amino acid sequence shown in SEQ ID NO: 1, and / or has higher thermal stability compared to the fructosyl amino acid oxidase containing the amino acid sequence shown in SEQ ID NO: 1, wherein the thermal stability is stability to a temperature of 45°C.

5. The fructosyl amino acid oxidase according to claim 4, which satisfies at least (2) and (3) above.

6. The fructosyl amino acid oxidase according to claim 4, wherein at least (2) and (3) above are satisfied, and (2) is such that when aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 71 of SEQ ID NO: 1 is selected from arginine (R), lysine (K), and histidine (H).

7. The fructosyl amino acid oxidase according to claim 6, further satisfying the following (10): (10) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 58 of SEQ ID NO: 1 is phenylalanine (F).

8. Furthermore, the fructosyl amino acid oxidase according to claim 7, which satisfies (1) above.

9. Furthermore, a fructosyl amino acid oxidase according to any one of claims 5 to 7, which satisfies (6) and (7) above.

10. A fructosyl amino acid oxidase having an amino acid sequence shown in any of Sequence IDs 2 to 278, or an amino acid sequence in which 1 to 15 amino acids are deleted, substituted, added and / or inserted in the above amino acid sequence, and which satisfies one or more of the following (1) to (9): (1) When aligned with the amino acid sequence shown in Sequence ID 1, the amino acid corresponding to position 35 of Sequence ID 1 is aspartic acid (D). (2) When aligned with the amino acid sequence shown in Sequence ID 1, the amino acid corresponding to position 71 of Sequence ID 1 is selected from arginine (R), lysine (K), histidine (H), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (3) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 101 of SEQ ID NO: 1 is selected from phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), histidine (H), glutamic acid (E), and isoleucine (I). (4) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 117 of SEQ ID NO: 1 is serine (S). (5) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 203 of SEQ ID NO: 1 is valine (V). (6) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 216 of SEQ ID NO: 1 is histidine (H). (7) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 217 of SEQ ID NO: 1 is arginine (R). (8) When aligned with the amino acid sequence shown in Sequence ID No. 1, the amino acid corresponding to position 247 of Sequence ID No. 1 is selected from glutamic acid (E), arginine (R), glutamine (Q), tyrosine (Y), alanine (A), and phenylalanine (F). (9) When aligned with the amino acid sequence shown in Sequence ID No. 1, the amino acid corresponding to position 417 of Sequence ID No. 1 is serine (S).

11. The fructosyl amino acid oxidase according to claim 10, further satisfying the following (10): (10) When aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 58 of SEQ ID NO: 1 is phenylalanine (F).

12. A gene encoding the amino acid sequence of the fructosyl amino acid oxidase according to claim 1 or 10.

13. An expression vector comprising the gene described in claim 12.

14. A glycated protein sensor comprising: a fructosyl amino acid oxidase according to claim 1 or 10; and a support on which the fructosyl amino acid oxidase is immobilized.

15. A method for measuring glycated proteins by the reaction of fructosyl amino acid oxidase as described in claim 1 or 10.

16. A method for producing fructosyl amino acid oxidase, comprising an amino acid substitution step, in which, when the amino acid sequence of fructosyl amino acid oxidase is aligned with the amino acid sequence shown in Sequence ID No. 1, an amino acid corresponding to a position selected from the group consisting of positions 35, 71, 101, 117, 203, 216, 217, 247, and 417 in the amino acid sequence shown in Sequence ID No. 1 is substituted.

17. The method for producing fructosyl amino acid oxidase according to claim 16, wherein the amino acid sequence of the fructosyl amino acid oxidase is an amino acid sequence having 95% or more homology with the amino acid sequence shown in Sequence ID No. 1.

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