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

Abstract

[Problem] In a glycated protein sensor including an immobilized enzyme, it is essential to develop a technique for enhancing the accuracy of measurement and the stability of the glycated protein sensor. However, an enzyme capable of enhancing the accuracy or stability of measurement using glycosylated protein sensors is not known. [Solution] The present invention provides a fructosyl amino acid oxidase having a residual activity of at least 70% after being treated at 65-80 °C for 20 minutes while being fixed to a support. In addition, the present invention provides a glycated protein sensor and a method for measuring a glycated protein.

Description

Fructosyl amino acid oxidase, method for producing fructosyl amino acid oxidase, glycated protein sensor, and method for measuring glycated protein.

[0001] The present invention relates to fructosyl amino acid oxidase, a method for producing fructosyl amino acid oxidase, a glycated protein sensor comprising fructosyl amino acid oxidase immobilized on a support, and a method for measuring glycated proteins, in which glycated proteins are detected by the reaction of fructosyl amino acid oxidase immobilized on a support.

[0002] Glycated proteins are measured as indicators for diagnosing diabetes and managing blood glucose control. For 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 enzymatic methods.

[0003] The enzymatic method for measuring glycated proteins involves, in a first step, decomposing proteins into amino acids or peptides using protease; in a second step, fructosyl amino acid oxidase is allowed to act on glycated amino acids (hereinafter also referred to as "glycated amino acids" or "fructosyl amino acids") among the amino acids or peptides, or peptides containing glycated amino acids (hereinafter also referred to as "glycated peptides" or "fructosyl peptides"), to generate hydrogen peroxide; and in a third step, the hydrogen peroxide is converted into a color-developing reaction and the absorbance is measured, or electrons released by the decomposition of hydrogen peroxide at an electrode are detected (see Patent Document 1).

[0004] In order to accurately measure specific glycated amino acids or glycated peptides, known inventions relate to modified fructosyl amino acid oxidases, and reagents and methods for measuring glycated proteins that have characteristics of fructosyl amino acid oxidases. For example, there is a fructosyl amino acid oxidase having an amino acid sequence obtained by modifying the amino acid sequence of a fructosyl amino acid oxidase found in the genus Coniochaeta, which acts on fructosyl valyl histidine but does not substantially act on other glycated amino acids (see Patent Documents 2 and 3), a method for measuring hemoglobin A1c by a reaction using a fructosyl amino acid oxidase that satisfies the property that its reactivity with fructosyl lysine is 30% or less of its reactivity with fructosyl valyl histidine (see Patent Document 4), and a method for measuring hemoglobin A1c by a reaction using a fructosyl amino acid oxidase that satisfies the property that its reactivity with fructosyl lysine is 12 or less, where the activity value with fructosyl valyl histidine is taken as 100. Known examples of such a fructosyl amino acid oxidase include a reagent composition for measuring glycated proteins, which comprises a fructosyl amino acid oxidase having the desired properties (see Patent Document 5); a reagent composition for measurement, which comprises an enzyme derived from a fructosyl amino acid oxidase of the genus Coniochaeta, etc., and which has a reactivity with fructosyl valine of 5% or less when the reactivity with fructosyl lysine is taken as 100% (see Patent Document 6); a method for measuring glycated hemoglobin, which comprises a fructosyl amino acid oxidase derived from the genus Coniochaeta or a modified enzyme thereof, which has a significantly reduced reactivity with fructosyl lysine relative to the reactivity with fructosyl valyl histidine over a wide pH range (see Patent Document 7); and a method for measuring fructosyl lysine using a fructosyl amino acid oxidase from Aspergillus oryzae, which acts on fructosyl lysine but does not substantially act on fructosyl valine (see Patent Document 8).

[0005] International Publication No. 2019 / 221264, Japanese Patent Application Laid-Open No. 2014-183786, International Publication No. 2012 / 018094, Japanese Patent Application Laid-Open No. 2011-229526, Japanese Patent Application Laid-Open No. 2010-233502, Japanese Patent No. 6504586, Japanese Patent Application Laid-Open No. 2013-176351, Japanese Patent Application Laid-Open No. 2009-000084

[0006] A first object of the present invention is to provide a novel fructosyl amino acid oxidase.

[0007] A second object of the present invention is to provide a fructosyl amino acid oxidase having excellent physicochemical properties.

[0008] A third object of the present invention is to provide an enzyme that contributes to the accuracy and stability of measurement in a glycated protein sensor having an immobilized enzyme.

[0009] A fourth object of the present invention is to provide a glycated protein sensor comprising a novel fructosyl amino acid oxidase and / or a method for measuring glycated proteins using the sensor.

[0010] The present inventors have discovered novel fructosyl amino acid oxidases from genes of unknown function, and have further discovered that some of these fructosyl amino acid oxidases have excellent physicochemical properties, and have succeeded in imparting these properties to other fructosyl amino acid oxidases.

[0011] Specifically, the present invention provides a wild-type fructosyl amino acid oxidase. Another aspect of the present invention provides a fructosyl amino acid oxidase having an amino acid sequence modified to impart excellent physicochemical properties in the measurement of glycated proteins, and a method for producing the same. Yet another aspect of the present invention provides a glycated protein sensor comprising the fructosyl amino acid oxidase and a method for measuring glycated proteins.

[0012] That is, the fructosyl amino acid oxidase of the present invention is a fructosyl amino acid oxidase that satisfies one or more selected from the following (1) to (5): (1) It comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, substituted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 1. (2) It comprises an amino acid sequence that has 30% or more homology with the amino acid sequence described in (1) above and satisfies the following (2-1) and (2-2): (2-1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid in the amino acid sequence corresponding to positions 247 and / or 277 in the amino acid sequence shown in SEQ ID NO: 1 is lysine. (2-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid in the amino acid sequence corresponding to positions 430 and / or 443 in the amino acid sequence shown in SEQ ID NO: 1 is lysine. (3) It comprises an amino acid sequence of any of SEQ ID NOs: 2 to 23, or an amino acid sequence in which one or several amino acids are deleted, substituted, added, and / or inserted in the amino acid sequence. (4) The residual activity after treatment at 48°C when immobilized on a support is higher than the residual activity after treatment under the same conditions when not immobilized on a support. (5) The residual activity after treatment at 65-80°C for 20 minutes when immobilized on a support is 70% or more.

[0013] The present invention may also provide a fructosyl amino acid oxidase that satisfies one or more of the conditions (1) to (3) and also satisfies the conditions (4) and / or (5).

[0014] Furthermore, the present invention may be directed to a fructosyl amino acid oxidase (4) and / or (5) that is immobilized on a support by crosslinking with an amine-reactive crosslinking agent.

[0015] The fructosyl amino acid oxidase of the present invention may be a recombinant protein.

[0016] Another aspect of the present invention is a fructosyl amino acid oxidase that satisfies the above-mentioned condition (2) and further satisfies one or more of the following conditions (2-3) to (2-4): (2-3) when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid in the amino acid sequence corresponding to positions 309 and / or 413 in the amino acid sequence shown in SEQ ID NO: 1 is lysine; and (2-4) when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid in the amino acid sequence corresponding to position 424 in the amino acid sequence shown in SEQ ID NO: 1 is lysine.

[0017] Another aspect of the present invention is a fructosyl amino acid oxidase that satisfies the above-mentioned condition (2) and further satisfies one or more of the following conditions (2-5) to (2-6): (2-5) when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids corresponding to positions 424, 430, and 443 in the amino acid sequence shown in SEQ ID NO: 1 are lysine; and (2-6) when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids corresponding to positions 309 and 413 in the amino acid sequence shown in SEQ ID NO: 1 are lysine.

[0018] Furthermore, the present invention may further provide a fructosyl amino acid oxidase that satisfies the above-mentioned (4).

[0019] Furthermore, in the present invention, the (4) may be fructosyl amino acid oxidase immobilized on a support by crosslinking with an amine-reactive crosslinking agent.

[0020] The amino acid sequence of the fructosyl amino acid oxidase of the present invention exhibits high homology to the amino acid sequence shown in SEQ ID NO: 1, specifically, it can be 74% or more.

[0021] Another aspect of the present invention is a fructosyl amino acid oxidase that satisfies all of the above items (2-3) to (2-4), and further, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids at positions 34, 177, 233, 234, and 297 in the amino acid sequence shown in SEQ ID NO: 1 are lysines.

[0022] The amino acid sequence of the fructosyl amino acid oxidase of the present invention exhibits high homology to the amino acid sequence shown in SEQ ID NO: 1, specifically, it can be 74% or more.

[0023] Another aspect of the present invention is a fructosyl amino acid oxidase that has a homology of 30% or more with the amino acid sequence shown in SEQ ID NO: 1, and that, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, contains a lysine at the amino acid position 247 and / or the amino acid position 277 in the amino acid sequence shown in SEQ ID NO: 1, and that further satisfies the following (6-1) and / or (6-2): (6-1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids in the amino acid sequence corresponding to positions 64, 84, 87, 128, 157, 166, 167, 394, and 397 in the amino acid sequence shown in SEQ ID NO: 1 are lysine. (6-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids in the amino acid sequence corresponding to positions 112, 137, 152, 247, 254, 255, 267, 277, and 438 in the amino acid sequence shown in SEQ ID NO: 1 are lysine.

[0024] Another aspect of the present invention is a fructosyl amino acid oxidase having a homology of 30% or more with the amino acid sequence shown in SEQ ID NO: 1, comprising 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 at position 247 and / or 277 in the amino acid sequence shown in SEQ ID NO: 1 is lysine, and further having the amino acid substitutions described in (7-1) and / or (7-2) below: (7-1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, at a position which is lysine in the amino acid sequence shown in SEQ ID NO: 1 but is an amino acid other than lysine in the amino acid sequence shown in SEQ ID NO: 1, the non-lysine amino acid is substituted with lysine; and (7-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, at a position which is an amino acid other than lysine in the amino acid sequence shown in SEQ ID NO: 1 but is lysine in the amino acid sequence shown in SEQ ID NO: 1, the lysine is substituted with the amino acid at that position in the amino acid sequence shown in SEQ ID NO: 1.

[0025] The fructosyl amino acid oxidase of the present invention may be a fructosyl amino acid oxidase that has a higher residual activity after heat treatment in a support-immobilized state, compared to the fructosyl amino acid oxidases comprising the amino acid sequences before amino acid substitution described in (7-1) and (7-2) above.

[0026] The fixation to the support may be achieved by crosslinking with an amine-reactive crosslinking agent, and the heat treatment may be performed at 48°C to 80°C.

[0027] In the fructosyl amino acid oxidase of the present invention, the positions in (7-1) and (7-2) may be one or more selected from positions 56 to 99, 120 to 132, 155 to 171, and 353 to 404 in the amino acid sequence shown in SEQ ID NO: 1; positions 100 to 119, 133 to 154, 241 to 288, and 435 to 440 in the amino acid sequence shown in SEQ ID NO: 1; and positions 1 to 55, 172 to 240, 289 to 352, 405 to 434, and 441 to 446 in the amino acid sequence shown in SEQ ID NO: 1.

[0028] The fructosyl amino acid oxidase of the present invention may be a fructosyl amino acid oxidase having an amino acid sequence having 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and comprising an amino acid sequence that satisfies one or more of the following (a1) to (a6) when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1: (a1) the amino acid at the position corresponding to position 61 of SEQ ID NO: 1 is arginine; (a2) the amino acid at the position corresponding to position 67 of SEQ ID NO: 1 is glutamic acid; (a3) ​​the amino acid at the position corresponding to position 128 of SEQ ID NO: 1 is lysine; (a4) the amino acid at the position corresponding to position 157 of SEQ ID NO: 1 is lysine; (a5) the amino acid at the position corresponding to position 166 of SEQ ID NO: 1 is lysine; and (a6) the amino acid at the position corresponding to position 391 of SEQ ID NO: 1 is histidine.

[0029] Another fructosyl amino acid oxidase of the present invention may be a fructosyl amino acid oxidase having an amino acid sequence that has 30% or more homology with the amino acid sequence of SEQ ID NO: 1, and that, when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 1, contains an amino acid sequence that satisfies one or more of the following (b1) to (b7): (b1) the amino acid at position 112 of SEQ ID NO: 1 is lysine; (b2) the amino acid at position 115 of SEQ ID NO: 1 is glutamic acid; (b3) the amino acid at position 137 of SEQ ID NO: 1 is lysine; (b4) the amino acid at position 247 of SEQ ID NO: 1 is lysine; (b5) the amino acid at position 254 of SEQ ID NO: 1 is lysine; (b6) the amino acid at position 277 of SEQ ID NO: 1 is lysine; and (b7) the amino acid at position 438 of SEQ ID NO: 1 is lysine.

[0030] Another fructosyl amino acid oxidase of the present invention may be a fructosyl amino acid oxidase having an amino acid sequence that has 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and that, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, contains an amino acid sequence that satisfies one or more of the following (c1) to (c11): (c1) The amino acid at the position corresponding to position 34 of SEQ ID NO:1 is lysine. (c2) The amino acid at the position corresponding to position 37 of SEQ ID NO:1 is threonine. (c3) The amino acid at the position corresponding to position 177 of SEQ ID NO:1 is lysine. (c4) The amino acid at the position corresponding to position 198 of SEQ ID NO:1 is glutamic acid. (c5) The amino acid at the position corresponding to position 297 of SEQ ID NO:1 is lysine. (c6) The amino acid at the position corresponding to position 302 of SEQ ID NO:1 is valine. (c7) The amino acid at the position corresponding to position 413 of SEQ ID NO:1 is lysine. (c8) The amino acid at the position corresponding to position 424 of SEQ ID NO:1 is lysine. (c9) The amino acid at the position corresponding to position 430 of SEQ ID NO:1 is lysine. (c10) The amino acid at the position corresponding to position 432 of SEQ ID NO:1 is glycine. (c11) The amino acid at the position corresponding to position 443 of SEQ ID NO:1 is lysine.

[0031] Another fructosyl amino acid oxidase of the present invention may be a fructosyl amino acid oxidase having an amino acid sequence that has 30% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and that, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, contains an amino acid sequence that satisfies one or more of the following (a1) to (a6), (b1) to (b7), and (c1) to (c11):(a1) The amino acid at the position corresponding to position 61 of SEQ ID NO: 1 is arginine. (a2) The amino acid at the position corresponding to position 67 of SEQ ID NO: 1 is glutamic acid. (a3) ​​The amino acid at the position corresponding to position 128 of SEQ ID NO: 1 is lysine. (a4) The amino acid at the position corresponding to position 157 of SEQ ID NO: 1 is lysine. (a5) The amino acid at the position corresponding to position 166 of SEQ ID NO: 1 is lysine. (a6) The amino acid at the position corresponding to position 391 of SEQ ID NO: 1 is histidine. (b1) The amino acid at the position corresponding to position 112 of SEQ ID NO: 1 is lysine. (b2) The amino acid at the position corresponding to position 115 of SEQ ID NO: 1 is glutamic acid. (b3) The amino acid at the position corresponding to position 137 of SEQ ID NO: 1 is lysine. (b4) The amino acid at the position corresponding to position 247 of SEQ ID NO: 1 is lysine. (b5) The amino acid at the position corresponding to position 254 of SEQ ID NO: 1 is lysine. (b6) (b7) The amino acid at the position corresponding to position 277 of SEQ ID NO:1 is lysine. (c1) The amino acid at the position corresponding to position 34 of SEQ ID NO:1 is lysine. (c2) The amino acid at the position corresponding to position 37 of SEQ ID NO:1 is threonine. (c3) The amino acid at the position corresponding to position 177 of SEQ ID NO:1 is lysine. (c4) The amino acid at the position corresponding to position 198 of SEQ ID NO:1 is glutamic acid. (c5) The amino acid at the position corresponding to position 297 of SEQ ID NO:1 is lysine. (c6) The amino acid at the position corresponding to position 302 of SEQ ID NO:1 is valine. (c7) The amino acid at the position corresponding to position 413 of SEQ ID NO:1 is lysine. (c8) The amino acid at the position corresponding to position 424 of SEQ ID NO:1 is lysine. (c9) The amino acid at the position corresponding to position 430 of SEQ ID NO:1 is lysine. (c10) The amino acid at the position corresponding to position 432 of SEQ ID NO:1 is glycine. (c11) The amino acid at the position corresponding to position 443 of SEQ ID NO: 1 is lysine.

[0032] The fructosyl amino acid oxidase of the present invention may have a reactivity specific to fructosyl lysine. The reactivity may be such that the reactivity to fructosyl valine or a peptide containing fructosyl valine is 20 or less when the reactivity to fructosyl lysine is set to 100. Furthermore, the reactivity may be such that the reactivity to fructosyl glycine is 20 or less when the reactivity to fructosyl lysine is set to 100.

[0033] The present invention also provides a method for producing a fructosyl amino acid oxidase, which method comprises substituting, with lysine, amino acids in an amino acid sequence of a fructosyl amino acid oxidase having 30% or more homology with the amino acid sequence of SEQ ID NO: 1 that correspond to one or more amino acids selected from positions 309, 413, 424, 430, and 443 of the amino acid sequence of SEQ ID NO: 1 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 1, wherein the thermostability of the fructosyl amino acid oxidase comprising the amino acid sequence after the amino acid substitutions when immobilized on a support is higher than the thermostability of the fructosyl amino acid oxidase comprising the amino acid sequence before the amino acid substitutions when immobilized on a support.

[0034] Another method for producing a fructosyl amino acid oxidase includes substituting any of the following amino acids (8-1) to (8-3) with lysine in an amino acid sequence of a fructosyl amino acid oxidase having 30% or more homology to the amino acid sequence set forth in SEQ ID NO: 1, wherein the thermostability of the fructosyl amino acid oxidase comprising the amino acid sequence after the amino acid substitution when immobilized on a support is higher than the thermostability of the fructosyl amino acid oxidase comprising the amino acid sequence before the amino acid substitution when immobilized on a support. (8-1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids on the amino acid sequence correspond to positions 64, 84, 87, 128, 157, 166, 167, 394, and 397 of the amino acid sequence shown in SEQ ID NO: 1. (8-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids on the amino acid sequence correspond to positions 112, 137, 152, 247, 254, 255, 267, 277, and 438 of the amino acid sequence shown in SEQ ID NO: 1. (8-3) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids on the amino acid sequence correspond to positions 34, 177, 233, 234, 297, 309, 413, 424, 430, and 443 of the amino acid sequence shown in SEQ ID NO: 1.

[0035] Another method for producing a fructosyl amino acid oxidase includes the steps of making the following amino acid substitutions (9-1) and (9-2) in an amino acid sequence of a fructosyl amino acid oxidase having 30% or more homology to the amino acid sequence shown in SEQ ID NO: 1, wherein the thermostability of the fructosyl amino acid oxidase comprising the amino acid sequence after the amino acid substitutions when immobilized on a support is higher than the thermostability of the fructosyl amino acid oxidase comprising the amino acid sequence before the amino acid substitutions when immobilized on a support: (9-1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, at a position which is lysine in the amino acid sequence shown in SEQ ID NO: 1 but is an amino acid other than lysine in the amino acid sequence, the non-lysine amino acid is substituted with lysine; and (9-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, at a position which is an amino acid other than lysine in the amino acid sequence shown in SEQ ID NO: 1 but is lysine in the amino acid sequence, the lysine is substituted with the amino acid at that position in the amino acid sequence shown in SEQ ID NO: 1.

[0036] The thermostability may be thermostability in a state where the fructosyl amino acid oxidase is immobilized on the support by crosslinking with an amine-reactive crosslinker. Furthermore, the residual activity of the fructosyl amino acid oxidase produced by the production method of the present invention after treatment at 48°C in a state where the fructosyl amino acid oxidase is immobilized on a support may be higher than the residual activity after treatment under the same conditions in a state where the fructosyl amino acid oxidase is not immobilized on a support, and / or the residual activity after treatment at 65°C to 80°C for 20 minutes in a state where the fructosyl amino acid oxidase is immobilized on a support may be 70% or more. The amino acid sequence homology in the production method of the present invention may be 74% or more.

[0037] The present invention also provides a glycated protein sensor comprising a support and fructosyl amino acid oxidase immobilized on the support, wherein the fructosyl amino acid oxidase, while immobilized on the support, has a residual activity of 70% or more after treatment at 65°C to 80°C for 20 minutes. The fructosyl amino acid oxidase may be immobilized on the support by crosslinking with an amine-reactive crosslinker.

[0038] The present invention also provides a glycated protein sensor comprising a support and fructosyl amino acid oxidase immobilized on the support, wherein the fructosyl amino acid oxidase is the fructosyl amino acid oxidase described above. The fructosyl amino acid oxidase may be immobilized on the support by crosslinking with an amine-reactive crosslinker.

[0039] The present invention also provides a glycated protein sensor further comprising a hydrogen peroxide detection unit. In the glycated protein sensor, a support layer containing a support and the hydrogen peroxide detection unit may be laminated.

[0040] Furthermore, the present invention provides a method for measuring glycated proteins, in which glycated proteins are detected by the reaction of fructosyl amino acid oxidase immobilized on a support.

[0041] The fructosyl amino acid oxidase of the present invention has specific reactivity to a specific glycated amino acid and / or a specific glycated peptide among glycated amino acids and / or glycated peptides, and by using the fructosyl amino acid oxidase of the present invention, the amount of a specific glycated amino acid and / or a specific glycated peptide contained in a test sample can be accurately measured.

[0042] Furthermore, according to the present invention, the thermal stability of fructosyl amino acid oxidase immobilized on a support can be increased, thereby improving the long-term stability of a glycated protein sensor containing the enzyme. The glycated protein sensor of the present invention is advantageous in that it is possible to obtain highly reliable measurement results over a long period of time because the decrease in enzyme activity during storage is suppressed.

[0043] 1 is a diagram showing the results of multiple alignment analysis of the amino acid sequences of fructosyl amino acid oxidases of Examples and Comparative Examples. FIG. 2 is a diagram showing the results of multiple alignment analysis of the amino acid sequences of fructosyl amino acid oxidases of Examples and Comparative Examples. FIG. 3 is a schematic diagram of a glycated protein sensor 10 according to one embodiment of the present invention. FIG. 4 is a schematic diagram of a glycated protein sensor 20 according to another embodiment of the present invention. FIG. 5 is a diagram showing the results of SDS-PAGE of the fructosyl amino acid oxidase of the Examples. FIG. 6 is a diagram showing the results of evaluating the thermal stability of the fructosyl amino acid oxidase of the Examples in a liquid phase. FIG. 7 is a diagram showing the results of evaluating the thermal stability of the fructosyl amino acid oxidase of the Examples when immobilized on a support. FIG. 8 is a diagram showing the results of evaluating the thermal stability of the fructosyl amino acid oxidase of the Examples when immobilized on a support. FIG. 9 is a diagram showing the results of evaluating the thermal stability of the fructosyl amino acid oxidase of the Examples and modified fructosyl amino acid oxidase when immobilized on a support.

[0044] The fructosyl amino acid oxidase of the present invention is a novel fructosyl amino acid oxidase. The novel fructosyl amino acid oxidase of the present invention includes a fructosyl amino acid oxidase with high thermostability. Furthermore, the present invention provides a method for producing a fructosyl amino acid oxidase modified to have increased thermostability. Furthermore, the present invention provides a glycated protein sensor including a support and fructosyl amino acid oxidase immobilized on the support, and a method for measuring glycated proteins, in which glycated proteins are detected by the reaction of the fructosyl amino acid oxidase immobilized on the support. The present invention will be described in detail below based on embodiments.

[0045] Fructosyl amino acid oxidase is an enzyme that acts on amino acids whose α-amino group and / or ε-amino group is 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.

[0046] Fructosyl amino acid oxidase uses glycated amino acids and / or glycated peptides as substrates. Specific examples of glycated amino acids include ε-fructosyl lysine (sometimes referred to as "fructosyl lysine"), α-fructosyl valine (sometimes referred to as "fructosyl valine"), α-fructosyl glycine (sometimes referred to as "fructosyl glycine"), α-fructosyl histididine (sometimes referred to as "fructosyl histidine"), α-fructosyl leucine (sometimes referred to as "fructosyl leucine"), and α-fructosyl serine (sometimes referred to as "fructosyl serine").

[0047] Specific examples of glycated 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 containing one or more glycated amino acids, such as α-fructosyl valyl histidine (sometimes referred to as "fructosyl valyl histidine").

[0048] The fructosyl amino acid oxidase of the present invention has the following physicochemical properties: an optimum pH range of 7 to 9, a working pH range of 5 to 9, a working temperature range of 20 to 80°C, and is soluble in a buffer solution. Specific examples of the buffer solution include Tris-hydrochloride buffer and phosphate-buffered saline (PBS). Furthermore, the molecular weight of the fructosyl amino acid oxidase of the present invention on SDS-PAGE may be about 45 kDa to about 55 kDa, and preferably about 48 kDa to about 50 kDa.

[0049] Furthermore, in one embodiment of the present invention, the fructosyl amino acid oxidase is heat-stable when immobilized on a support. The fructosyl amino acid oxidase that is heat-stable when immobilized on a support is a fructosyl amino acid oxidase 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 70% or more residual activity after heat treatment at 65°C or higher.

[0050] More specifically, the treatment temperature may be 48° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., or 95° C. The treatment time is 10 to 30 minutes, preferably 15 to 25 minutes, and most preferably 20 minutes.

[0051] Another example of a fructosyl amino acid oxidase that is heat-stable when immobilized on a support is a fructosyl amino acid oxidase whose residual activity after heat treatment when immobilized on a support is higher than the residual activity after heat treatment under the same conditions when not immobilized on a support. More specifically, the treatment temperature is 48°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C. The treatment time is 10 to 30 minutes, preferably 15 to 25 minutes, and most preferably 20 minutes.

[0052] To confirm the thermal stability of fructosyl amino acid oxidase, the support onto which fructosyl amino acid oxidase is immobilized may be heated using a known method such as an electric oven or a thermostatic bath, and the residual activity of the fructosyl amino acid oxidase may be measured. Here, the residual activity refers to the ratio of the enzyme activity after heat treatment at each temperature to the enzyme activity before heat treatment.

[0053] Examples of means for immobilizing an enzyme such as fructosyl amino acid oxidase include covalent bonding, physical adsorption, ionic bonding, cross-linking, entrapment, biochemical specific binding, etc. Depending on the enzyme to be used, an immobilization method that does not inactivate the enzyme may be selected, or a combination of multiple immobilization methods may be used.

[0054] In some embodiments, the enzyme is immobilized on the support by crosslinking with a crosslinker, either alone or in a state bound to a carrier. In such cases, the support is a protein different from the enzyme, and the enzyme and support are mixed together, and then the enzyme is immobilized on the support using a crosslinker such as glutaraldehyde or an isocyanate derivative. Specific examples of proteins include albumins such as bovine serum albumin (BSA), collagen, and gelatin. The support may also be the same enzyme as the enzyme to be immobilized. In such cases, also known as self-aggregation, the enzyme is immobilized on the same enzyme by crosslinking with a crosslinker.

[0055] The crosslinking agent used is a substance that crosslinks between molecules or within molecules, and specific examples thereof include glutaraldehyde, isocyanate derivatives, formaldehyde, glyoxal, malondialdehyde, and succinaldehyde. Among these, amine-reactive crosslinking agents are preferably used. Amine-reactive crosslinking agents react with amino groups in proteins to crosslink the proteins, and specific examples thereof include glutaraldehyde, formaldehyde, N-hydroxyesters, amide esters, and imide esters.

[0056] The amine-reactive crosslinker reacts with amino groups present on the surface of the fructosyl amino acid oxidase to crosslink and immobilize the fructosyl amino acid oxidase to the support. The fructosyl amino acid oxidase may be heat stable in a state immobilized on the support.

[0057] In other embodiments, the support may be a synthetic polymer, a resin, an inorganic material, a polysaccharide, a mineral, a clay, etc. Specific examples include: when a fluororesin, an ion exchange resin, a polyvinyl alcohol resin, a hydraulic resin, a photocurable resin, a solid polymer electrolyte, a polyion complex, a urethane, etc. is used as the support, the enzyme is immobilized on the support by chemical interactions such as hydrophobicity; when charcoal, bone charcoal, silica gel, glass, zeolite, celite, alumina, titanium oxide, ceramic, hydroxyapatite, etc. is used as the support, the enzyme is immobilized on the support by physical interactions between the support and the enzyme; when a semipermeable membrane such as nylon, cellophane, ethyl cellulose, acetyl cellulose, polystyrene, or a phospholipid membrane is used as the support, the enzyme is immobilized on the support by encapsulating the enzyme in the membrane; and when a polyacrylamide gel, agar, gelatin, carrageenan, sodium alginate gel, calcium alginate gel, chitosan gel, etc. is used as the support, the enzyme is immobilized on the gel.

[0058] The support may also be beads. The beads may be made of carbon particles (carbon beads), silica (SiO ２ ) Microparticles (silica beads), beads made of polysaccharides such as chitin, chitosan, and alginic acid, may also be used. 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 and 200 nm or less. The enzyme may be immobilized by being crosslinked to the beads.

[0059] The fructosyl amino acid oxidase of the present invention may specifically react with a specific glycated amino acid or a peptide containing that amino acid. In one embodiment, the fructosyl amino acid oxidase has high specificity for fructosyl lysine. "High specificity for fructosyl lysine" means that, when the reactivity toward fructosyl lysine or a peptide containing fructosyl lysine is taken as 100, the reactivity toward other glycated amino acids or peptides containing other glycated amino acids is 20 or less. Here, the reactivity toward 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" refers to, for example, one or more selected from fructosyl valine, fructosyl glycine, fructosyl histidine, fructosyl leucine, and fructosyl serine.

[0060] Fructosyl amino acid oxidases are classified into three groups based on substrate specificity. Fructosyl amino acid oxidases belonging to Group 1 are highly specific for amino acids whose α-amino group is glycated and / or peptides containing such amino acids. Fructosyl amino acid oxidases belonging to Group 2 are highly specific for amino acids whose ε-amino group is glycated and / or peptides containing such amino acids. Fructosyl amino acid oxidases belonging to Group 3 are highly specific for amino acids whose α-amino group is glycated and / or peptides containing such amino acids, as well as amino acids whose ε-amino group is glycated and / or peptides containing such amino acids. The fructosyl amino acid oxidase of the present invention may belong to any of the groups.

[0061] In another embodiment, the fructosyl amino acid oxidase has high specificity for fructosyl valyl histidine. "High specificity for fructosyl valyl histidine" means that, when the reactivity towards fructosyl valyl histidine is taken as 100, the reactivity towards other glycated amino acids or other glycated peptides is 20 or less. Here, the reactivity towards 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" refers to, for example, one or more selected from fructosyl valine, fructosyl lysine, fructosyl glycine, fructosyl histidine, fructosyl leucine, and fructosyl serine, and "other fructosyl peptides" refers to peptides containing glycated amino acids other than fructosyl valyl histidine.

[0062] The fructosyl amino acid oxidase of the present invention may specifically react with a specific glycated amino acid or a peptide containing that amino acid. In one embodiment, the fructosyl amino acid oxidase has high specificity for fructosyl valine. "High specificity for fructosyl valine" means that, when the reactivity toward fructosyl valine or a peptide containing fructosyl valine is taken as 100, the reactivity toward other glycated amino acids or peptides containing other glycated amino acids is 20 or less. Here, the reactivity toward 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" refers to, for example, one or more selected from fructosyl lysine, fructosyl glycine, fructosyl histidine, fructosyl leucine, and fructosyl serine.

[0063] The fructosyl amino acid oxidase of the present invention may be a recombinant protein expressed by introducing a nucleic acid encoding the fructosyl amino acid oxidase of the present invention into a host such as Escherichia coli, fungi, yeast, mammalian cells, insect cells, etc. Furthermore, the fructosyl amino acid oxidase may be not only one cloned from a microorganism, animal, plant, etc., but also one artificially mutated, modified, or designed.

[0064] The present inventors have discovered novel fructosyl amino acid oxidases from genes of unknown function. Among these, a fructosyl amino acid oxidase was found to have significantly high thermostability when immobilized on a support. Based on the amino acid sequence of this fructosyl amino acid oxidase, they created a variant of another fructosyl amino acid oxidase and succeeded in increasing its thermostability. The amino acid sequence of the novel fructosyl amino acid oxidase discovered by the present inventors is shown in Table 1.

[0065]

[0066] Fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1 is derived from Aspergillus pseudotamarii, a species of the Aspergillus genus. Based on sequence homology, it was previously predicted to be a type of FAD-dependent oxidoreductase [Accession Number: XP_031920077], but it was not known that such a protein could function as a fructosyl amino acid oxidase. The present inventors have discovered that fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1 has remarkably high thermostability when immobilized on a support and extremely high substrate specificity for specific glycated amino acids and / or glycated peptides.

[0067] Fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 2 is derived from Penicillium rubens of the genus Penicillium. Although it was previously known as a hypothetical protein [Accession Number: XP_002568014], it was not known that this protein could function as a fructosyl amino acid oxidase. The present inventors have discovered that the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 2 has extremely high substrate specificity for specific glycated amino acids and / or glycated peptides.

[0068] Fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 3 is derived from Penicillium flavigenum of the genus Penicillium. Although it has been known as a hypothetical protein [Accession Number: OQE32666], it was not previously known that this protein could function as a fructosyl amino acid oxidase. The present inventors have discovered that the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 3 has extremely high substrate specificity for specific glycated amino acids and / or glycated peptides.

[0069] The fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 4 is derived from Monascus purpureus of the genus Monascus. Although it has been known as a hypothetical protein [Accession Number: TQB73262], it was not previously known that this protein could function as a fructosyl amino acid oxidase. The present inventors have discovered that the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 4 has extremely high substrate specificity for specific glycated amino acids and / or glycated peptides.

[0070] The fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 5 is derived from Coniochaeta pulveracea of ​​the genus Coniochaeta. Although it has been known as a hypothetical protein [Accession Number: RKU49498], it was not previously known that this protein could function as a fructosyl amino acid oxidase. The present inventors have discovered that the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 5 has extremely high substrate specificity for specific glycated amino acids and / or glycated peptides.

[0071] The present invention also provides a fructosyl amino acid oxidase having an amino acid sequence highly homologous to any of the amino acid sequences of SEQ ID NOs: 1 to 5, which fructosyl amino acid oxidase has physicochemical properties such as high thermostability when immobilized on a support and high substrate specificity for specific glycated amino acids or glycated peptides. Specifically, the amino acid sequence of the fructosyl amino acid oxidase may include an amino acid sequence 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, 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, or 80% or more identity to the amino acid sequence of SEQ ID NO: 1. These homology values ​​are rounded to one decimal place.

[0072] In another embodiment of the present invention, the fructosyl amino acid oxidase included in the glycated protein sensor may be a fructosyl amino acid oxidase having an amino acid sequence other than that of SEQ ID NO: 1 or SEQ ID NO: 1. The fructosyl amino acid oxidase having an amino acid sequence other than that of SEQ ID NO: 1 may be a modified fructosyl amino acid oxidase that has been imparted with physicochemical properties such as high thermostability when immobilized on a support and high substrate specificity for specific glycated amino acids or glycated peptides.

[0073] As will be described later, it has been found that the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1 has extremely high heat stability when immobilized on a support, and exhibits excellent physicochemical properties, such as a residual activity of 70% or more after treatment at 65°C to 80°C for 20 minutes when immobilized on a support, and / or a residual activity after heat treatment when immobilized on a support that is higher than the residual activity after treatment under the same conditions when not immobilized on a support.

[0074] Furthermore, the inventors have examined the amino acid sequence shown in SEQ ID NO: 1 in detail and have found that the lysines at positions 247 and / or 277, and 430 and / or 443 of the amino acid sequence shown in SEQ ID NO: 1 play important roles in the thermostability of the enzyme.

[0075] That is, one embodiment of the present invention is a fructosyl amino acid oxidase consisting of an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NO: 1 and satisfying the following requirements (1) and (2), or may also be a fructosyl amino acid oxidase comprising such an amino acid sequence: (1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid in the amino acid sequence corresponding to positions 247 and / or 277 in the amino acid sequence shown in SEQ ID NO: 1 is lysine; (2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid in the amino acid sequence corresponding to positions 430 and / or 443 in the amino acid sequence shown in SEQ ID NO: 1 is lysine.

[0076] Here, the results of multiple alignment analysis of the amino acid sequences of known fructosyl amino acid oxidases, SEQ ID NOs: 1 to 5, as well as SEQ ID NOs: 24 and 25, are shown in Figures 1 and 2. Here, SEQ ID NO: 24 is a fructosyl amino acid oxidase derived from Aspergillus oryzae [Accession Number: BAD54824], and SEQ ID NO: 25 is a fructosyl amino acid oxidase derived from Aspergillus fumigatus [Accession Number: AAB88209]. In the figures, the amino acids at positions 247, 277, 430, and 443 in the amino acid sequence set forth in SEQ ID NO: 1 are indicated by arrows. Furthermore, the amino acids in SEQ ID NOs: 2 to 5, SEQ ID NO: 24, and SEQ ID NO: 25 corresponding to the lysines at positions 247, 277, 430, and 443 in the amino acid sequence set forth in SEQ ID NO: 1 are shown in Table 2. As shown in Table 2, the amino acid sequences that satisfy the above (1) and (2) are distinguished from the amino acid sequences of SEQ ID NOs: 24 and 25, which are known fructosyl amino acid oxidases.

[0077] In another embodiment of the present invention, the fructosyl amino acid oxidase is an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NO: 1, wherein, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids at positions 247, 277, 430, and 443 in the amino acid sequence shown in SEQ ID NO: 1 are lysines. Alternatively, the fructosyl amino acid oxidase may comprise such an amino acid sequence.

[0078]

[0079]

[0023] Furthermore, it has been revealed that, in addition to the above-mentioned amino acids, lysines at positions 309, 413, and 424 in the amino acid sequence of SEQ ID NO: 1 also play important roles in the thermostability of the enzyme.

[0024] That is, one embodiment of the present invention relates to a fructosyl amino acid oxidase having an amino acid sequence highly homologous to the amino acid sequence of SEQ ID NO: 1, such that, when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 1, the amino acids at positions 309, 413, and / or 424 in the amino acid sequence of SEQ ID NO: 1 are lysines, or the present invention may also provide a fructosyl amino acid oxidase comprising such an amino acid sequence. In Figure 2, the amino acids at positions 309, 413, and 424 in the amino acid sequence of SEQ ID NO: 1 are indicated by arrowheads.

[0080] In another embodiment of the present invention, the fructosyl amino acid oxidase is an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NO: 1, wherein, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids at positions 309, 413, and / or 443 in the amino acid sequence shown in SEQ ID NO: 1 are lysines. Alternatively, the fructosyl amino acid oxidase may comprise such an amino acid sequence.

[0081] Furthermore, it has been revealed that, in addition to the above-mentioned amino acids, lysines at positions 34, 177, 233, 234, and 297 in the amino acid sequence of SEQ ID NO: 1 also play important roles in the thermostability of the enzyme. That is, one embodiment of the present invention relates to a fructosyl amino acid oxidase having an amino acid sequence highly homologous to the amino acid sequence of SEQ ID NO: 1, such that, when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 1, the amino acids at positions 34, 177, 233, 234, and / or 297 in the amino acid sequence of SEQ ID NO: 1 are lysines, or the present invention may also provide a fructosyl amino acid oxidase comprising such an amino acid sequence. In Figures 1 and 2 , the amino acids at positions 34, 177, 233, 234, and 297 in the amino acid sequence of SEQ ID NO: 1 are indicated by white arrowheads.

[0082] In another embodiment of the present invention, the fructosyl amino acid oxidase is an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NO: 1, wherein, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids at positions 34, 177, 233, 234, and 297 in the amino acid sequence shown in SEQ ID NO: 1 are lysines. Alternatively, the fructosyl amino acid oxidase may comprise such an amino acid sequence.

[0083] Furthermore, in addition to the above-mentioned amino acids, it has been revealed that the lysines at positions 64, 84, 87, 128, 157, 166, 167, 394, and 397 in the amino acid sequence of SEQ ID NO: 1, as well as the lysines at positions 112, 137, 152, 247, 254, 255, 267, 277, and 438 also play important roles in the thermostability of the enzyme.

[0084] That is, in one embodiment of the present invention, there is provided a fructosyl amino acid oxidase having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NO: 1, which satisfies the following conditions (3-1) and / or (4-1), or the fructosyl amino acid oxidase may comprise such an amino acid sequence. (3-1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence contains lysine at one or more positions selected from 64, 84, 87, 128, 157, 166, 167, 394, and 397 in the amino acid sequence shown in SEQ ID NO: 1. (4-1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence contains lysine at one or more positions selected from 112, 137, 152, 247, 254, 255, 267, 277, and 438 in the amino acid sequence shown in SEQ ID NO: 1.

[0085] In another embodiment of the present invention, the fructosyl amino acid oxidase has an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NO: 1 and satisfies the requirements of (3-2) and / or (4-2) below. Alternatively, the fructosyl amino acid oxidase may comprise such an amino acid sequence. (3-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids in the amino acid sequence corresponding to positions 64, 84, 87, 128, 157, 166, 167, 394, and 397 in the amino acid sequence shown in SEQ ID NO: 1 are lysines. (4-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids in the amino acid sequence corresponding to positions 112, 137, 152, 247, 254, 255, 267, 277, and 438 in the amino acid sequence shown in SEQ ID NO: 1 are lysines.

[0086] The present invention also provides a method for producing a fructosyl amino acid oxidase comprising a modified amino acid sequence, which comprises substituting an amino acid on the amino acid sequence of the fructosyl amino acid oxidase. More specifically, this step may be a step of substituting, with lysine, one or more amino acids in the amino acid sequence before modification corresponding to positions 34, 64, 84, 87, 112, 177, 128, 137, 152, 157, 166, 167, 233, 234, 247, 254, 255, 267, 277, 297, 309, 394, 397, 413, 424, 430, 438, and 443 of SEQ ID NO: 1 when the amino acid sequence of a fructosyl amino acid oxidase having high homology to the amino acid sequence of SEQ ID NO: 1 (hereinafter referred to as the "amino acid sequence before modification") is aligned with the amino acid sequence of SEQ ID NO: 1.

[0087] It has been revealed that the above-mentioned amino acids in the amino acid sequence shown in SEQ ID NO: 1 play an important role in the thermostability of the enzyme. Therefore, the fructosyl amino acid oxidase modified by the production method of the present invention has higher thermostability than the fructosyl amino acid oxidase before modification. More preferably, the fructosyl amino acid oxidase modified by the production method of the present invention has higher thermostability when immobilized on a support than the fructosyl amino acid oxidase before modification.

[0088] The present inventors have found that the physicochemical properties of fructosyl amino acid oxidase can be improved by dividing SEQ ID NO: 1 into three regions [(a), (b), and (c)] based on the three-dimensional structure of fructosyl amino acid oxidase and modifying amino acids in each region, where region (a) consists of amino acids corresponding to positions 56 to 99, 120 to 132, 155 to 171, and 353 to 404 of SEQ ID NO: 1, region (b) consists of amino acids corresponding to positions 100 to 119, 133 to 154, 241 to 288, and 435 to 440 of SEQ ID NO: 1, and region (c) consists of amino acids corresponding to positions 1 to 55, 172 to 240, 289 to 352, 405 to 434, and 441 to 446 of SEQ ID NO: 1.

[0089] Another embodiment of the method for producing a fructosyl amino acid oxidase of the present invention comprises modifying amino acids in each region of a fructosyl amino acid oxidase having an amino acid sequence highly homologous to SEQ ID NO: 1 so that the following conditions (1) and (2) are satisfied. This production method makes it possible to obtain an enzyme with higher thermostability compared to the enzyme before modification. (1) When the amino acid sequence before modification is aligned with the amino acid sequence shown in SEQ ID NO: 1, at a position which is lysine in the amino acid sequence of SEQ ID NO: 1 but is an amino acid other than lysine in the amino acid sequence before modification, the non-lysine amino acid is substituted with lysine. (2) When the amino acid sequence before modification is aligned with the amino acid sequence shown in SEQ ID NO: 1, at a position which is an amino acid other than lysine in the amino acid sequence of SEQ ID NO: 1 but is lysine in the amino acid sequence before modification, the lysine is substituted with the amino acid other than lysine, i.e., the amino acid at that position in the amino acid sequence shown in SEQ ID NO: 1.

[0090] Here, alignment of amino acid sequences can be performed using programs such as Blast, Clustal W, Clustal Omega, etc. Alignment provides information such as homology, identity, or similarity between multiple amino acid sequences, and the positions of corresponding amino acids.

[0091] An example of a modified fructosyl amino acid oxidase obtained by the present invention is shown below. The fructosyl amino acid oxidase has been modified so that, when its amino acid sequence is aligned with the amino acid sequence set forth in SEQ ID NO: 1, it satisfies one or more of the following conditions (a1) to (a6), (b1) to (b7), and (c1) to (c11), and has improved thermal stability when immobilized on a support compared to the fructosyl amino acid oxidase before modification.(a1) The amino acid at the position corresponding to position 61 of SEQ ID NO: 1 is arginine. (a2) The amino acid at the position corresponding to position 67 of SEQ ID NO: 1 is glutamic acid. (a3) ​​The amino acid at the position corresponding to position 128 of SEQ ID NO: 1 is lysine. (a4) The amino acid at the position corresponding to position 157 of SEQ ID NO: 1 is lysine. (a5) The amino acid at the position corresponding to position 166 of SEQ ID NO: 1 is lysine. (a6) The amino acid at the position corresponding to position 391 of SEQ ID NO: 1 is histidine. (b1) The amino acid at the position corresponding to position 112 of SEQ ID NO: 1 is lysine. (b2) The amino acid at the position corresponding to position 115 of SEQ ID NO: 1 is glutamic acid. (b3) The amino acid at the position corresponding to position 137 of SEQ ID NO: 1 is lysine. (b4) The amino acid at the position corresponding to position 247 of SEQ ID NO: 1 is lysine. (b5) The amino acid at the position corresponding to position 254 of SEQ ID NO: 1 is lysine. (b6) (b7) The amino acid at the position corresponding to position 438 of SEQ ID NO:1 is lysine. (c1) The amino acid at the position corresponding to position 34 of SEQ ID NO:1 is lysine. (c2) The amino acid at the position corresponding to position 37 of SEQ ID NO:1 is threonine. (c3) The amino acid at the position corresponding to position 177 of SEQ ID NO:1 is lysine. (c4) The amino acid at the position corresponding to position 198 of SEQ ID NO:1 is glutamic acid. (c5) The amino acid at the position corresponding to position 297 of SEQ ID NO:1 is lysine. (c6) The amino acid at the position corresponding to position 302 of SEQ ID NO:1 is valine. (c7) The amino acid at the position corresponding to position 413 of SEQ ID NO:1 is lysine. (c8) The amino acid at the position corresponding to position 424 of SEQ ID NO:1 is lysine. (c9) The amino acid at the position corresponding to position 430 of SEQ ID NO:1 is lysine. (c10) The amino acid at the position corresponding to position 432 of SEQ ID NO:1 is glycine. (c11) The amino acid at the position corresponding to position 443 of SEQ ID NO: 1 is lysine.

[0092] Specific examples of amino acid sequences of modified fructosyl amino acid oxidases are shown below. The amino acid sequence shown in SEQ ID NO:6 is a modified version of the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence shown in SEQ ID NO:2 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:6. (a1) Lysine at position 61 of SEQ ID NO:2 (corresponding to position 61 of SEQ ID NO:1) was substituted with arginine. (a2) Lysine at position 67 of SEQ ID NO:2 (corresponding to position 67 of SEQ ID NO:1) was substituted with glutamic acid. (a3) ​​Alanine at position 128 of SEQ ID NO:2 (corresponding to position 128 of SEQ ID NO:1) was substituted with lysine. (a4) Arginine at position 157 of SEQ ID NO:2 (corresponding to position 157 of SEQ ID NO:1) was substituted with lysine. (a5) Arginine at position 166 of SEQ ID NO:2 (corresponding to position 166 of SEQ ID NO:1) was substituted with lysine. (a6) The lysine at position 391 of SEQ ID NO:2 (corresponding to position 391 of SEQ ID NO:1) was substituted with histidine.

[0093] The amino acid sequence shown in SEQ ID NO:7 is a modified version of the amino acid sequence shown in SEQ ID NO:3. The amino acid sequence shown in SEQ ID NO:3 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:7. (a1) Lysine at position 61 of SEQ ID NO:3 (corresponding to position 61 of SEQ ID NO:1) was substituted with arginine. (a2) Lysine at position 67 of SEQ ID NO:3 (corresponding to position 67 of SEQ ID NO:1) was substituted with glutamic acid. (a3) ​​Alanine at position 128 of SEQ ID NO:3 (corresponding to position 128 of SEQ ID NO:1) was substituted with lysine. (a4) Arginine at position 157 of SEQ ID NO:3 (corresponding to position 157 of SEQ ID NO:1) was substituted with lysine. (a5) Arginine at position 166 of SEQ ID NO:3 (corresponding to position 166 of SEQ ID NO:1) was substituted with lysine. (a6) Lysine at position 391 of SEQ ID NO:3 (corresponding to position 391 of SEQ ID NO:1) was substituted with histidine.

[0094] The amino acid sequence shown in SEQ ID NO:8 is a modified version of the amino acid sequence shown in SEQ ID NO:4. The amino acid sequence shown in SEQ ID NO:4 was aligned with the amino acid sequence shown in SEQ ID NO:1, and amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO:8. (a1) Lysine at position 60 of SEQ ID NO:4 (corresponding to position 61 of SEQ ID NO:1) was substituted with arginine. (a5) Arginine at position 159 of SEQ ID NO:4 (corresponding to position 166 of SEQ ID NO:1) was substituted with lysine.

[0095] The amino acid sequence shown in SEQ ID NO:9 is a modified version of the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in SEQ ID NO:5 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the amino acids were substituted as follows to obtain the amino acid sequence shown in SEQ ID NO:9. (a4) Proline at position 154 of SEQ ID NO:5 (corresponding to position 157 of SEQ ID NO:1) was substituted with lysine. (a5) Arginine at position 163 of SEQ ID NO:5 (corresponding to position 166 of SEQ ID NO:1) was substituted with lysine. (a6) Lysine at position 387 of SEQ ID NO:5 (corresponding to position 391 of SEQ ID NO:1) was substituted with histidine.

[0096] The amino acid sequence shown in SEQ ID NO:10 is a modified version of the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence shown in SEQ ID NO:2 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:10. (b1) Glutamic acid at position 112 of SEQ ID NO:2 (corresponding to position 112 of SEQ ID NO:1) was substituted with lysine. (b2) Lysine at position 115 of SEQ ID NO:2 (corresponding to position 115 of SEQ ID NO:1) was substituted with glutamic acid. (b3) Glutamine at position 137 of SEQ ID NO:2 (corresponding to position 137 of SEQ ID NO:1) was substituted with lysine. (b4) Arginine at position 247 of SEQ ID NO:2 (corresponding to position 247 of SEQ ID NO:1) was substituted with lysine. (b5) Aspartic acid at position 254 of SEQ ID NO:2 (corresponding to position 254 of SEQ ID NO:1) was substituted with lysine. (b7) Arginine at position 438 of SEQ ID NO:2 (corresponding to position 438 of SEQ ID NO:1) was substituted with lysine.

[0097] The amino acid sequence shown in SEQ ID NO:11 is a modified version of the amino acid sequence shown in SEQ ID NO:3. The amino acid sequence shown in SEQ ID NO:3 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:11. (b1) Glutamic acid at position 112 of SEQ ID NO:3 (corresponding to position 112 of SEQ ID NO:1) was substituted with lysine. (b2) Lysine at position 115 of SEQ ID NO:3 (corresponding to position 115 of SEQ ID NO:1) was substituted with glutamic acid. (b3) Glutamine at position 137 of SEQ ID NO:3 (corresponding to position 137 of SEQ ID NO:1) was substituted with lysine. (b4) Arginine at position 247 of SEQ ID NO:3 (corresponding to position 247 of SEQ ID NO:1) was substituted with lysine. (b5) Glutamic acid at position 254 of SEQ ID NO:3 (corresponding to position 254 of SEQ ID NO:1) was substituted with lysine. (b7) The serine at position 438 of SEQ ID NO: 3 (corresponding to position 438 of SEQ ID NO: 1) was substituted with lysine.

[0098] The amino acid sequence shown in SEQ ID NO: 12 is a modified version of the amino acid sequence shown in SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 4 was aligned with the amino acid sequence shown in SEQ ID NO: 1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO: 12. (b1) Glutamic acid at position 105 of SEQ ID NO: 4 (corresponding to position 112 of SEQ ID NO: 1) was substituted with lysine. (b7) Arginine at position 433 of SEQ ID NO: 4 (corresponding to position 438 of SEQ ID NO: 1) was substituted with lysine.

[0099] The amino acid sequence shown in SEQ ID NO:13 is a modified version of the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in SEQ ID NO:5 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:13. (b1) Glutamic acid at position 109 of SEQ ID NO:5 (corresponding to position 112 of SEQ ID NO:1) was substituted with lysine. (b3) Glycine at position 134 of SEQ ID NO:5 (corresponding to position 137 of SEQ ID NO:1) was substituted with lysine. (b4) Proline at position 244 of SEQ ID NO:5 (corresponding to position 247 of SEQ ID NO:1) was substituted with lysine. (b5) Arginine at position 251 of SEQ ID NO:5 (corresponding to position 254 of SEQ ID NO:1) was substituted with lysine. (b7) Asparagine at position 434 of SEQ ID NO:5 (corresponding to position 438 of SEQ ID NO:1) was substituted with lysine.

[0100] The amino acid sequence shown in SEQ ID NO:14 is a modified version of the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence shown in SEQ ID NO:2 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:14. (c1) Arginine at position 34 of SEQ ID NO:2 (corresponding to position 34 of SEQ ID NO:1) was substituted with lysine. (c2) Lysine at position 37 of SEQ ID NO:2 (corresponding to position 37 of SEQ ID NO:1) was substituted with threonine. (c3) Glutamine at position 177 of SEQ ID NO:2 (corresponding to position 177 of SEQ ID NO:1) was substituted with lysine. (c4) Lysine at position 198 of SEQ ID NO:2 (corresponding to position 198 of SEQ ID NO:1) was substituted with glutamic acid. (c5) Aspartic acid at position 297 of SEQ ID NO:2 (corresponding to position 297 of SEQ ID NO:1) was substituted with lysine. (c6) Lysine at position 302 of SEQ ID NO:2 (corresponding to position 302 of SEQ ID NO:1) was substituted with valine. (c8) Arginine at position 424 of SEQ ID NO:2 (corresponding to position 424 of SEQ ID NO:1) was substituted with lysine. (c9) Aspartic acid at position 430 of SEQ ID NO:2 (corresponding to position 430 of SEQ ID NO:1) was substituted with lysine. (c10) Lysine at position 432 of SEQ ID NO:2 (corresponding to position 432 of SEQ ID NO:1) was substituted with glycine. (c11) Methionine at position 443 of SEQ ID NO:2 (corresponding to position 443 of SEQ ID NO:1) was substituted with lysine.

[0101] The amino acid sequence shown in SEQ ID NO:15 is a modified version of the amino acid sequence shown in SEQ ID NO:3. The amino acid sequence shown in SEQ ID NO:3 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:15. (c2) Lysine at position 37 of SEQ ID NO:3 (corresponding to position 37 of SEQ ID NO:1) was substituted with threonine. (c3) Glutamine at position 177 of SEQ ID NO:3 (corresponding to position 177 of SEQ ID NO:1) was substituted with lysine. (c4) Lysine at position 198 of SEQ ID NO:3 (corresponding to position 198 of SEQ ID NO:1) was substituted with glutamic acid. (c5) Aspartic acid at position 297 of SEQ ID NO:3 (corresponding to position 297 of SEQ ID NO:1) was substituted with lysine. (c6) Lysine at position 302 of SEQ ID NO:3 (corresponding to position 302 of SEQ ID NO:1) was substituted with valine. (c8) Arginine at position 424 of SEQ ID NO:3 (corresponding to position 424 of SEQ ID NO:1) was substituted with lysine. (c9) Aspartic acid at position 430 of SEQ ID NO:3 (corresponding to position 430 of SEQ ID NO:1) was substituted with lysine. (c10) Lysine at position 432 of SEQ ID NO:3 (corresponding to position 432 of SEQ ID NO:1) was substituted with glycine. (c11) Methionine at position 443 of SEQ ID NO:3 (corresponding to position 443 of SEQ ID NO:1) was substituted with lysine.

[0102] The amino acid sequence shown in SEQ ID NO:16 is a modified version of the amino acid sequence shown in SEQ ID NO:4. The amino acid sequence shown in SEQ ID NO:4 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:16. (c5) Asparagine at position 292 of SEQ ID NO:4 (corresponding to position 297 of SEQ ID NO:1) was substituted with lysine. (c6) Lysine at position 297 of SEQ ID NO:4 (corresponding to position 302 of SEQ ID NO:1) was substituted with valine. (c9) Glutamic acid at position 425 of SEQ ID NO:4 (corresponding to position 430 of SEQ ID NO:1) was substituted with lysine. (c11) Serine at position 438 of SEQ ID NO:4 (corresponding to position 443 of SEQ ID NO:1) was substituted with lysine.

[0103] The amino acid sequence shown in SEQ ID NO:17 is a modified version of the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in SEQ ID NO:5 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:17. (c5) Glycine at position 294 of SEQ ID NO:5 (corresponding to position 297 of SEQ ID NO:1) was substituted with lysine. (c8) Glutamic acid at position 426 of SEQ ID NO:5 (corresponding to position 430 of SEQ ID NO:1) was substituted with lysine. (c10) Proline at position 439 of SEQ ID NO:5 (corresponding to position 443 of SEQ ID NO:1) was substituted with lysine.

[0104] The amino acid sequence shown in SEQ ID NO:18 is a modified version of the amino acid sequence shown in SEQ ID NO:2. The amino acid sequence shown in SEQ ID NO:2 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:18. (a1) Lysine at position 61 of SEQ ID NO:2 (corresponding to position 61 of SEQ ID NO:1) was substituted with arginine. (a2) Lysine at position 67 of SEQ ID NO:2 (corresponding to position 67 of SEQ ID NO:1) was substituted with glutamic acid. (a3) ​​Alanine at position 128 of SEQ ID NO:2 (corresponding to position 128 of SEQ ID NO:1) was substituted with lysine. (a4) Arginine at position 157 of SEQ ID NO:2 (corresponding to position 157 of SEQ ID NO:1) was substituted with lysine. (a5) Arginine at position 166 of SEQ ID NO:2 (corresponding to position 166 of SEQ ID NO:1) was substituted with lysine. (a6) A lysine at position 391 of SEQ ID NO:2 (corresponding to position 391 of SEQ ID NO:1) was substituted with histidine. (b1) A glutamic acid at position 112 of SEQ ID NO:2 (corresponding to position 112 of SEQ ID NO:1) was substituted with lysine. (b2) A lysine at position 115 of SEQ ID NO:2 (corresponding to position 115 of SEQ ID NO:1) was substituted with glutamic acid. (b3) A glutamine at position 137 of SEQ ID NO:2 (corresponding to position 137 of SEQ ID NO:1) was substituted with lysine. (b4) An arginine at position 247 of SEQ ID NO:2 (corresponding to position 247 of SEQ ID NO:1) was substituted with lysine. (b5) An aspartic acid at position 254 of SEQ ID NO:2 (corresponding to position 254 of SEQ ID NO:1) was substituted with lysine. (b7) An arginine at position 438 of SEQ ID NO:2 (corresponding to position 438 of SEQ ID NO:1) was substituted with lysine. (c1) Arginine at position 34 of SEQ ID NO:2 (corresponding to position 34 of SEQ ID NO:1) was substituted with lysine. (c2) Lysine at position 37 of SEQ ID NO:2 (corresponding to position 37 of SEQ ID NO:1) was substituted with threonine. (c3) Glutamine at position 177 of SEQ ID NO:2 (corresponding to position 177 of SEQ ID NO:1) was substituted with lysine. (c4) Lysine at position 198 of SEQ ID NO:2 (corresponding to position 198 of SEQ ID NO:1) was substituted with glutamic acid. (c5) Aspartic acid at position 297 of SEQ ID NO:2 (corresponding to position 297 of SEQ ID NO:1) was substituted with lysine. (c6) Lysine at position 302 of SEQ ID NO:2 (corresponding to position 302 of SEQ ID NO:1) was substituted with valine.(c8) Arginine at position 424 of SEQ ID NO:2 (corresponding to position 424 of SEQ ID NO:1) was substituted with lysine. (c9) Aspartic acid at position 430 of SEQ ID NO:2 (corresponding to position 430 of SEQ ID NO:1) was substituted with lysine. (c10) Lysine at position 432 of SEQ ID NO:2 (corresponding to position 432 of SEQ ID NO:1) was substituted with glycine. (c11) Methionine at position 443 of SEQ ID NO:2 (corresponding to position 443 of SEQ ID NO:1) was substituted with lysine.

[0105] The amino acid sequence shown in SEQ ID NO:19 is a modified version of the amino acid sequence shown in SEQ ID NO:3. The amino acid sequence shown in SEQ ID NO:3 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:19. (a1) Lysine at position 61 of SEQ ID NO:3 (corresponding to position 61 of SEQ ID NO:1) was substituted with arginine. (a2) Lysine at position 67 of SEQ ID NO:3 (corresponding to position 67 of SEQ ID NO:1) was substituted with glutamic acid. (a3) ​​Alanine at position 128 of SEQ ID NO:3 (corresponding to position 128 of SEQ ID NO:1) was substituted with lysine. (a4) Arginine at position 157 of SEQ ID NO:3 (corresponding to position 157 of SEQ ID NO:1) was substituted with lysine. (a5) Arginine at position 166 of SEQ ID NO:3 (corresponding to position 166 of SEQ ID NO:1) was substituted with lysine. (a6) A lysine at position 391 of SEQ ID NO:3 (corresponding to position 391 of SEQ ID NO:1) was substituted with histidine. (b1) A glutamic acid at position 112 of SEQ ID NO:3 (corresponding to position 112 of SEQ ID NO:1) was substituted with lysine. (b2) A lysine at position 115 of SEQ ID NO:3 (corresponding to position 115 of SEQ ID NO:1) was substituted with glutamic acid. (b3) A glutamine at position 137 of SEQ ID NO:3 (corresponding to position 137 of SEQ ID NO:1) was substituted with lysine. (b4) An arginine at position 247 of SEQ ID NO:3 (corresponding to position 247 of SEQ ID NO:1) was substituted with lysine. (b5) A glutamic acid at position 254 of SEQ ID NO:3 (corresponding to position 254 of SEQ ID NO:1) was substituted with lysine. (b7) A serine at position 438 of SEQ ID NO:3 (corresponding to position 438 of SEQ ID NO:1) was substituted with lysine. (c2) The lysine at position 37 of SEQ ID NO:3 (corresponding to position 37 of SEQ ID NO:1) was substituted with threonine. (c3) The glutamine at position 177 of SEQ ID NO:3 (corresponding to position 177 of SEQ ID NO:1) was substituted with lysine. (c4) The lysine at position 198 of SEQ ID NO:3 (corresponding to position 198 of SEQ ID NO:1) was substituted with glutamic acid. (c5) The aspartic acid at position 297 of SEQ ID NO:3 (corresponding to position 297 of SEQ ID NO:1) was substituted with lysine. (c6) The lysine at position 302 of SEQ ID NO:3 (corresponding to position 302 of SEQ ID NO:1) was substituted with valine. (c8) The arginine at position 424 of SEQ ID NO:3 (corresponding to position 424 of SEQ ID NO:1) was substituted with lysine.(c9) Aspartic acid at position 430 of SEQ ID NO: 3 (corresponding to position 430 of SEQ ID NO: 1) was substituted with lysine. (c10) Lysine at position 432 of SEQ ID NO: 3 (corresponding to position 432 of SEQ ID NO: 1) was substituted with glycine. (c11) Methionine at position 443 of SEQ ID NO: 3 (corresponding to position 443 of SEQ ID NO: 1) was substituted with lysine.

[0106] The amino acid sequence shown in SEQ ID NO:20 is a modified version of the amino acid sequence shown in SEQ ID NO:4. The amino acid sequence shown in SEQ ID NO:4 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:20. (a1) Lysine at position 60 of SEQ ID NO:4 (corresponding to position 61 of SEQ ID NO:1) was substituted with arginine. (a5) Arginine at position 159 of SEQ ID NO:4 (corresponding to position 166 of SEQ ID NO:1) was substituted with lysine. (b1) Glutamic acid at position 105 of SEQ ID NO:4 (corresponding to position 112 of SEQ ID NO:1) was substituted with lysine. (b6) Arginine at position 433 of SEQ ID NO:4 (corresponding to position 438 of SEQ ID NO:1) was substituted with lysine. (c5) Asparagine at position 292 of SEQ ID NO:4 (corresponding to position 297 of SEQ ID NO:1) was substituted with lysine. (c6) Lysine at position 297 of SEQ ID NO:4 (corresponding to position 302 of SEQ ID NO:1) was substituted with valine. (c9) Glutamic acid at position 425 of SEQ ID NO:4 (corresponding to position 430 of SEQ ID NO:1) was substituted with lysine. (c11) Serine at position 438 of SEQ ID NO:4 (corresponding to position 443 of SEQ ID NO:1) was substituted with lysine.

[0107] The amino acid sequence shown in SEQ ID NO:21 is a modified version of the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in SEQ ID NO:5 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:21. (a4) Proline at position 154 of SEQ ID NO:5 (corresponding to position 157 of SEQ ID NO:1) was substituted with lysine. (a5) Arginine at position 163 of SEQ ID NO:5 (corresponding to position 166 of SEQ ID NO:1) was substituted with lysine. (a6) Lysine at position 387 of SEQ ID NO:5 (corresponding to position 391 of SEQ ID NO:1) was substituted with histidine. (b1) Glutamic acid at position 109 of SEQ ID NO:5 (corresponding to position 112 of SEQ ID NO:1) was substituted with lysine. (b3) Glycine at position 134 of SEQ ID NO:5 (corresponding to position 137 of SEQ ID NO:1) was substituted with lysine. (b4) Proline at position 244 of SEQ ID NO:5 (corresponding to position 247 of SEQ ID NO:1) was substituted with lysine. (b5) Arginine at position 251 of SEQ ID NO:5 (corresponding to position 254 of SEQ ID NO:1) was substituted with lysine. (b7) Asparagine at position 434 of SEQ ID NO:5 (corresponding to position 438 of SEQ ID NO:1) was substituted with lysine. (c5) Glycine at position 294 of SEQ ID NO:5 (corresponding to position 297 of SEQ ID NO:1) was substituted with lysine. (c9) Glutamic acid at position 426 of SEQ ID NO:5 (corresponding to position 430 of SEQ ID NO:1) was substituted with lysine. (c11) Proline at position 439 of SEQ ID NO:5 (corresponding to position 443 of SEQ ID NO:1) was substituted with lysine.

[0108] The SEQ ID NOs of the amino acid sequences of the fructosyl amino acid oxidase before and after modification, as well as the amino acids substituted in the amino acid sequence after modification and their positions are shown in Tables 3 to 6. For example, substitution of arginine (R) at position 34 with lysine (K) is represented as "R34K."

[0109]

[0110]

[0111]

[0112]

[0113] The amino acid sequence shown in SEQ ID NO:22 is a modified version of the amino acid sequence (SEQ ID NO:24) of a known FAOD derived from Aspergillus oryzae [Accession Number: BAD54824]. The amino acid sequence shown in SEQ ID NO:24 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:22. (b4) Arginine at position 247 of SEQ ID NO:24 (corresponding to position 247 of SEQ ID NO:1) was substituted with lysine (R247K). (b6) Asparagine at position 277 of SEQ ID NO:24 (corresponding to position 277 of SEQ ID NO:1) was substituted with lysine (N277K).

[0114] The amino acid sequence shown in SEQ ID NO:23 is a modified version of the amino acid sequence (SEQ ID NO:25) of a known FAOD derived from Aspergillus fumigatus [Accession Number: AAB88209]. The amino acid sequence shown in SEQ ID NO:25 was aligned with the amino acid sequence shown in SEQ ID NO:1, and the following amino acid substitutions were made to obtain the amino acid sequence shown in SEQ ID NO:23. (b4) Glutamine at position 247 of SEQ ID NO:25 (corresponding to position 247 of SEQ ID NO:1) was substituted with lysine (Q247K). (c7) Arginine at position 413 of SEQ ID NO:25 (corresponding to position 413 of SEQ ID NO:1) was substituted with lysine (R413K). (c8) Arginine at position 424 of SEQ ID NO:25 (corresponding to position 424 of SEQ ID NO:1) was substituted with lysine (R424K). (c9) Glutamine at position 430 of SEQ ID NO:25 (corresponding to position 430 of SEQ ID NO:1) was substituted with lysine (Q430K). (c11) Arginine at position 443 of SEQ ID NO:25 (corresponding to position 443 of SEQ ID NO:1) was substituted with lysine (R443K).

[0115] The fructosyl amino acid oxidase in which the amino acids have been modified as described above has improved thermal stability when immobilized on a support, compared to the fructosyl amino acid oxidase before modification.

[0116] In the present invention, a recombinant fructosyl amino acid oxidase protein consisting of these amino acid sequences can be used. Specifically, the fructosyl amino acid oxidase that can be used in the present invention can be obtained by culturing a host such as a yeast such as Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris, a mammalian cell, or an insect cell, into which an expression vector containing a base sequence encoding these amino acid sequences has been introduced, and then separating the host from the fungi or cells, purifying the protein, concentrating it, or the like.

[0117] Another embodiment of the present invention is a fructosyl amino acid oxidase comprising 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 set forth in any of SEQ ID NOS: 1 to 23, and which has physicochemical properties such as high thermostability when immobilized on a support and high substrate specificity for specific glycated amino acids or glycated peptides. Here, "one or several amino acids" refers to 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 amino acids.

[0118] The present invention further provides a glycated protein sensor. The glycated protein sensor of the present invention includes 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 can 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.

[0119] 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 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 proteins from the detection result of the detection unit.

[0120] 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 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.

[0121] In another embodiment, the detection unit is an optical detection unit, and hydrogen peroxide is detected by measuring absorbance or light intensity. For example, hydrogen peroxide is quantified by detecting a color reaction in the presence of peroxidase and an oxidative color-developing dye, or by detecting the luminescence intensity of luminol.

[0122] In another embodiment, 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 luminescence from a luminescent reagent such as luminol. The detection unit may be a hydrogen peroxide detection unit using a different method.

[0123] 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 diluted solution 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 body fluid-derived solution and a non-body fluid-derived solution. 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 solution.

[0124] The body fluid may be blood, serum, plasma, lymph, tissue fluid such as interstitial fluid, intercellular fluid, or interstitial fluid, or may be body cavity fluid, serous cavity fluid, pleural fluid, peritoneal fluid, pericardial fluid, cerebrospinal fluid (spinal fluid), joint fluid (synovial fluid), or aqueous humor (aqueous humor). The body fluid may be digestive fluid such as saliva, gastric juice, bile, pancreatic juice, or intestinal fluid, or may be sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, or milk. The body fluid may be animal body fluid or human body fluid. The body fluid may be liquid in foods containing animal-derived protein (e.g., milk or dairy products). The body fluid may be plant body fluid, plant biofluid, or plant-derived liquid. For example, the body fluid may be plant juice, nectar, or sap.

[0125] The solution may contain a substance to be measured. For example, the solution may be tears, and the substance to be measured may be albumin or glycated albumin contained in tears. Alternatively, the substance to be measured may be albumin, glycated albumin, hemoglobin, or glycated hemoglobin in blood, serum, or plasma, albumin or glycated albumin in interstitial fluid, albumin or glycated albumin in urine, or albumin or glycated albumin in saliva.

[0126] The measurement target of the glycated protein sensor of the present invention is a 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.

[0127] In one embodiment of the present invention, in addition to fructosyl amino acid oxidase, 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. Proteases may also be enzymes that decompose proteins into peptide fragments. When a protein contains a glycated amino acid residue, the action of the protease may produce one or more selected from the group consisting of glycated amino acids, peptide fragments containing glycated amino acids, non-glycated amino acids, and peptide fragments not containing glycated amino acids. Fructosyl amino acid oxidase may react with glycated amino acids or peptide fragments containing glycated amino acids to produce hydrogen peroxide.

[0128] In one embodiment of the present invention, the support to which the enzyme is immobilized forms a support layer such as a thin film layer, and is arranged by being laminated on 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.

[0129] Various bonding agents may be used as long as they do not substantially impede the measurement principle of the present disclosure. For example, the bonding agent may contain a material that bonds an inorganic material with an organic material. The bonding agent may be, for example, a silane coupling agent. Examples of silane coupling agents include the following: Vinyl-based: vinyltrimethoxysilane, vinyltriethoxysilane, 7-octenyltrimethoxysilane, vinyldimethylethoxysilane, vinylmethyldimethoxysilane, vinylmethyldiethoxysilane, vinyltris(2-methoxyethoxy)silane, vinyltris(trimethylsiloxy)silane, 4-vinylphenyltrimethoxysilane, allyltrimethoxysilane, allyltriethoxysilane, 5-(triethoxysilyl)-2-norbornene; Styryl-based: p-styryltrimethoxysilane; Methacrylic-based: 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltris(trimethylsiloxy)silane, 3-methacryloxypropyltriallylsilane, 8-methacryloxyoctyltrimethoxysilane; Acrylic: 3-acryloxypropyltrimethoxysilane, 3-acryloxypropyldimethylmethoxysilane, 3-acryloxypropyltriallylsilane; Epoxy: 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 8-glycidoxyoctyltrimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane;Amino-based: N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltriethoxysilane, N-2-(aminoethyl)-8-aminooctyltrimethoxysilane, N-6-(aminohexyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropylmethyldimethoxysilane, 3-aminopropyltriethoxysilane Triethoxysilane (APTES), 3-aminopropylmethyldiethoxysilane, 3-triethoxysilyl-N-(1,3-dimethyl-butylidene)propylamine, N-phenyl-3-aminopropyltrimethoxysilane, N-methyl-3-aminopropyltrimethoxysilane, N,N-dimethyl-3-aminopropyltrimethoxysilane, bis[3-(trimethoxysilyl)-propyl]amine, N-(vinylbenzyl)-2-aminoethyl-3-aminopropyltrimethoxysilane hydrochloride; Ureido-based: 3-ureidopropyltrimethoxysilane, 3-ureidopropyltriethoxysilane; Azide-based: 11-azidoundecyltrimethoxysilane; Isocyanate-based: 3-isocyanatepropyltrimethoxysilane, 3-isocyanatepropyltriethoxysilane; Isocyanurate-based: tris-(trimethoxysilylpropyl)isocyanurate; Mercapto-based: 3-mercaptopropyltrimethoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltriethoxysilane; Or, Acid anhydride: 3-trimethoxysilylpropylsuccinic anhydride.

[0130] Furthermore, the present invention provides a method for measuring glycated proteins, which is a method for measuring glycated proteins, in which glycated proteins are detected by a reaction of fructosyl amino acid oxidase immobilized on a support.

[0131] Figure 3 shows the configuration of a glycated protein sensor 10 according to one embodiment of the present invention. The sensor 10 shown in Figure 3 includes 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 cross-linked to bovine serum albumin, which serves as a support, with glutaraldehyde, which serves as a cross-linking agent. The bovine serum albumin is also cross-linked to itself with glutaraldehyde. These components form the enzyme layer 12. The enzyme layer 12 is laminated on 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 with a silane coupling agent 18.

[0132] In another embodiment, the glycated protein sensor further includes another layer in addition to the enzyme layer and the hydrogen peroxide detection unit. The other layer may be, for example, a limiting permeation layer, which may include polycarbonate or an ion exchange resin. For example, the limiting permeation layer may be provided outside the enzyme layer or between the enzyme layer and the hydrogen peroxide detection unit, and may be bonded by hydrogen bonds, ionic bonds, or the like.

[0133] The ion exchange resin may be a cation exchange resin and / or an anion exchange resin. For example, when a cation exchange resin such as Nafion (registered trademark) is used, anions present in the analyte can be suppressed or prevented from reaching the detection unit. For example, when an anion exchange resin such as polypyrrole is used, cations present in the analyte can be suppressed or prevented from reaching the detection unit. The permeation-limiting layer may contain one, multiple, or at least one type of ion exchange resin, and may be configured with one, multiple, or at least one type of layer.

[0134] When a glycated protein to be measured is introduced, the glycated protein is decomposed by the protease immobilized on the support, generating glycated and non-glycated peptide fragments. These peptide fragments are thought to diffuse within the enzyme layer 12, and when the glycated peptide fragments react with the immobilized fructosyl amino acid oxidase, hydrogen peroxide is generated.

[0135] The hydrogen peroxide detector 14 detects this hydrogen peroxide and outputs a signal related to its concentration. When the hydrogen peroxide detector 14 is a hydrogen peroxide electrode, hydrogen peroxide is decomposed at the hydrogen peroxide electrode, and the released electrons are detected as a current.

[0136] The glycated protein sensor 10 has a control unit that calculates the concentration of glycated protein from the current detected at the hydrogen peroxide electrode. The control unit can calculate the concentration of glycated protein in the sample solution from a predetermined relationship between the concentration of glycated protein and the current value generated at the hydrogen peroxide electrode.

[0137] Since fructosyl amino acid oxidase has high thermal stability when immobilized on the support body, bovine serum albumin, and / or has a reactivity specific to fructosyl lysine, the glycated protein sensor 10 can accurately calculate the concentration of a specific glycated protein.

[0138] Another embodiment of the present invention is shown in Fig. 4. In a glycated protein sensor 20, a layer containing a support on which fructosyl amino acid oxidase has been immobilized (fructosyl amino acid oxidase layer 22) is formed on the hydrogen peroxide detection unit 14. A layer containing a support on which protease has been immobilized (protease layer 24) is formed on this fructosyl amino acid oxidase layer 22, i.e., on the surface opposite to the hydrogen peroxide detection unit 14. In other words, the fructosyl amino acid oxidase layer 22 and the protease layer 24 are laminated in this order on the hydrogen peroxide detection unit 14.

[0139] In another embodiment, the glycated protein sensor further comprises another layer in addition to the enzyme layer and the hydrogen peroxide detection unit. The other layer may be, for example, the aforementioned permeation-restricting layer, which is provided on the outside of the fructosyl amino acid oxidase layer 22, between the fructosyl amino acid oxidase layer 22 and the protease layer 24, or between the fructosyl amino acid oxidase layer 22 and the hydrogen peroxide detection unit 14 and may be bonded by hydrogen bonding, ionic bonding, or the like.

[0140] When a glycated protein to be measured is introduced, the glycated protein is decomposed by the protease in the protease layer 24, generating glycated and non-glycated peptide fragments. These peptide fragments are thought to permeate the fructosyl amino acid oxidase layer 22, and when the glycated peptide fragments react with the immobilized fructosyl amino acid oxidase, hydrogen peroxide is generated, and the hydrogen peroxide detection unit 14 detects the generated hydrogen peroxide.

[0141] In another embodiment of the present invention, the glycated protein sensor has a fructosyl amino acid oxidase layer but does not have a protease immobilized on a support. A solution that has been pre-treated with a protease may be introduced into the glycated protein sensor as a measurement target, and hydrogen peroxide produced in the fructosyl amino acid oxidase layer may be detected by a hydrogen peroxide detection unit.

[0142] The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0143] The detailed method for producing the fructosyl amino acid oxidase used in this example is described below. First, a gene containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NOs: 1 to 5 and designed to add a histidine tag to the N-terminus of each inserted protein was introduced into an expression vector having a lactose operon. Escherichia coli BL21(DE3) strain (Novagen, Merck) was transformed with the expression vector. Hereinafter, the fructosyl amino acid oxidases consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 5 produced from the E. coli are referred to as Examples 1 to 5, respectively.

[0144] First, it was confirmed by SDS-PAGE that the target protein could be produced from E. coli into which a base sequence encoding a fructosyl amino acid oxidase consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 3 and SEQ ID NO: 5 had been introduced.

[0145] Each strain of E. coli was cultured with shaking for 2.5 hours in LB liquid medium containing 50 mg / mL kanamycin (Nacalai Tesque). Furthermore, each strain of E. coli was cultured with shaking overnight in LB liquid medium supplemented with isopropyl β-D-thiogalactopyranoside (hereinafter referred to as "IPTG") to a final concentration of 0.5 mM, and in LB liquid medium without IPTG. After cultivation, OD600 was measured to confirm the growth of E. coli.

[0146] The culture medium was centrifuged to obtain a bacterial cell pellet, to which cell lysis buffer (containing 98% by volume of HEPES buffer, 1% by volume of 100 mg / mL lysozyme solution, 1% by volume of 10% Triton (registered trademark) X-100 solution, and supplemented with 1% by volume of 0.5 M tris(2-carboxyethyl)phosphine and 0.2% by volume of 10 U / μL DpnI) was added so as to maintain a constant bacterial cell density, and the mixture was incubated on ice for 1 hour. The supernatant was collected by centrifugation, and the precipitate was mixed with HEPES buffer.

[0147] To 8 μL each of the supernatant and precipitate, 8 μL of sample buffer (2ME+) (x2) (Fujifilm Wako Pure Chemical Industries, Ltd.) was added and mixed, followed by heat treatment at 95°C for 10 minutes. The mixture was applied to an SDS-PAGE gel (Perfect NT Gel M, DRC Corporation), electrophoresed at a constant voltage of 180 V, and stained with Quick CBB Plus (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0148] The results are shown in Figure 5. In Figure 5, (A) shows the results of electrophoresis of the supernatant, and (B) shows the results of electrophoresis of the precipitate. In the figure, "M" indicates a molecular weight marker, and the numbers in each lane indicate the sequence number of the protein introduced into E. coli. "IPTG(-)" indicates E. coli cultured without addition of IPTG, and "IPTG(+)" indicates E. coli cultured with addition of IPTG. Proteins of approximately 48 to 53 kDa were present in the supernatant and precipitate of the bacterial cells treated with the cell lysate to which IPTG had been added, confirming that the recombinant fructosyl amino acid oxidases of Examples 1 to 3 and 5 had been produced.

[0149] Furthermore, the introduced protein was purified from the transformed E. coli. Each E. coli strain was cultured using the above-mentioned LB medium, and after expression induction with IPTG, culture was continued at 18°C ​​for 20 hours. The collected E. coli cells were suspended in buffer A (20 mM Tris-HCl (pH 7.5) containing 0.25 M sodium chloride and 20 mM imidazole), disrupted using an ultrasonicator, and centrifuged to obtain a cell lysate supernatant. The cell lysate supernatant was filtered through a 0.45 μm pore size filter and then applied to a HisTrap HP (GE Healthcare). The filter was washed with 6 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).

[0150] The resulting eluate was dialyzed against 20 mM potassium phosphate buffer (pH 7.5) and sterilized with a 0.22 μm pore size filter to obtain a sample. The protein concentration in the sample was measured using Protein Assay Kit I (Bio-Rad). The volume of the samples, including those in Examples 1 to 5, and the protein concentration in the samples are shown in Table 7.

[0151]

[0152] The fructosyl amino acid oxidase activity of Examples 1 to 5 was examined. As comparative examples, fructosyl amino acid oxidase FAOD-E (manufactured by Kikkoman Corporation, Comparative Example 1), Lucica® GA-L (manufactured by Asahi Kasei Pharma Corporation, Comparative Example 2), and a fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 26 (Comparative Example 3) were used. SEQ ID NO: 26 is known to be an FAD-dependent oxidoreductase derived from Ascosphaera apis [Accession Number: KZZ92706] and has 55 to 63% homology with the amino acid sequence of known fructosyl amino acid oxidases. The fructosyl amino acid oxidase of Comparative Example 1 was purified in the same manner as in Examples 1 to 5, and 10 mL of a protein solution with a concentration of 15.8 mg / mL was obtained.

[0153] The substrates used were fructosyl lysine (F-Lys), fructosyl valyl histidine (F-Val-His), fructosyl valine (F-Val), and fructosyl glycine (F-Gly). In Examples 1 to 5 and Comparative Examples 1 and 3, 20 μL of a substrate solution containing 1 mM substrate was added to each well of a microplate at room temperature. Then, 10 μL of a 0.5 wt% oxidative color-developing reagent, TOOS (Dojindo Research Institute), 260 μL of a solution containing 4.76 μg / mL peroxidase and 0.1 mg / mL 4-aminoantipyrine (Nacalai Tesque), HEPES buffer (pH 8.0) containing 0.15 M sodium chloride, and 5 μL of an appropriately diluted fructosyl amino acid oxidase solution were added and mixed. The change in absorbance at 555 nm was measured at 37°C. As a negative control, 20 mM potassium phosphate buffer (pH 7.5) was used.

[0154] In Comparative Example 2, 240 μL of pretreatment solution (GA R-1) was added to the well, followed by 6 μL of substrate. After reacting for 5 minutes at 37°C, 60 μL of enzyme solution (GA R-2) was added, and the reaction was continued for an additional 5 minutes at 37°C. Using purified water as a control, absorbance was measured at a dominant wavelength of 546 nm and a subordinate wavelength of 700 nm immediately before and 5 minutes after the addition of the enzyme solution, and the change in absorbance during this period was obtained.

[0155] In the Examples and Comparative Examples, fructosyl amino acid oxidase activity was confirmed when F-Lys was used as a substrate, and therefore the activity for each of F-Val-His, F-Val, and F-Gly was measured under the same conditions as for F-Lys. The activity for F-Lys and the specific activity for F-Val-His, F-Val, and F-Gly for the Examples and Comparative Examples are shown in Tables 8 and 9. High substrate specificity for F-Lys was confirmed in Examples 1 to 5.

[0156]

[0157]

[0158] The thermal stability in the liquid phase and in the immobilized state of the fructosyl amino acid oxidase (Example 1) consisting of the amino acid shown in SEQ ID NO: 1 purified by the above-mentioned method and FAOD-E (manufactured by Kikkoman Corporation, Comparative Example 1) as a comparative example were examined.

[0159] First, the liquid-phase thermal stability of the enzyme of Example 1 was compared with that of the enzyme of Comparative Example 1. 80 μL of HEPES buffer solution containing 1 U / mL of each enzyme (hereinafter referred to as "enzyme dilution") was dispensed into tubes and maintained on a heat block at temperatures of 30°C, 40°C, 50°C, and 55°C for 15 minutes. Next, 10 μL of 0.5 wt% TOOS, 270 μL of POD / 4-AA solution (diluted with HEPES buffer (pH 8.0, 0.15 M NaCl) to give 4.76 μg / mL peroxidase and 0.1 mg / mL 4-aminoantipyrine), and 5 μL of the heat-treated enzyme dilution were added to wells of a microtiter plate containing 20 μL of 1 mM F-Lys at room temperature, and the mixture was mixed by pipetting. Using a microtiter plate reader set at 37°C, absorbance at 555 nm was measured every 15 seconds for 10 minutes after addition, and the change in absorbance per unit time was taken as the enzyme activity. The enzyme activity at 30°C was taken as 100%, and the ratio of the activity at 30°C to the activity at 40°C, 50°C, and 55°C was calculated as the residual activity.

[0160] Furthermore, to clarify the thermal stability of the enzyme immobilized on the support, a glycated protein sensor (Example) was fabricated. A platinum electrode was subjected to silane coupling treatment to introduce amino groups onto the electrode surface. Furthermore, bovine serum albumin (BSA) was added to a TES buffer solution, and the enzyme from Example 1 was added and stirred. A predetermined amount of glutaraldehyde solution was then added to the mixture of enzyme and BSA and stirred. 1 μL of this solution was dropped onto the platinum electrode with the amino groups introduced, and the mixture was thoroughly dried to obtain the Example sensor. In the Comparative Example, the enzyme from Comparative Example 1 (FAOD-E, manufactured by Kikkoman Corporation) was used instead of the enzyme from Example 1.

[0161] 200 μL of HEPES buffer containing 50 μM F-Lys was dropped onto the sensors of the Examples and Comparative Examples, and current output values ​​were obtained. Each sensor was immersed in HEPES buffer heated to 50°C, 65°C, or 80°C and allowed to stand at that temperature for 20 minutes. Thereafter, to eliminate the influence of residual heat, each sensor was allowed to stand at 4°C for 30 minutes. Power output values ​​for F-Lys were obtained in the same manner as in the measurement before the temperature treatment. The power output value before the temperature treatment was set to 100%, and the ratio of the power output value after the temperature treatment to the power output value before the temperature treatment was calculated as the residual activity.

[0162] The results of measuring the thermal stability in the liquid phase are shown in Figure 6, and the results of measuring the thermal stability in the immobilized state are shown in Figure 7. The fructosyl amino acid oxidase of Example 1 exhibited residual activity in the liquid phase that was almost equivalent to that of the comparative example. On the other hand, when the enzyme was immobilized on a support, it exhibited residual activity of 70% or more with F-Lys even after treatment at 65°C or 80°C for 20 minutes, which was superior to that of the comparative example.

[0163] Next, the thermal stability of the modified fructosyl amino acid oxidases was examined. A fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 18 (hereinafter referred to as Example 6) and a fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 19 (hereinafter referred to as Example 7) were used. For comparison of thermal stability, a fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 2 (Example 2) and a fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 3 (Example 3) were used.

[0164] The method for producing the fructosyl amino acid oxidase in each example is described below. The nucleotide sequence encoding each amino acid sequence was introduced into the expression vector pET28b (Novagen, Merck), and Escherichia coli BL21(DE3) strain (Nippon Gene) was transformed. The strain was then inoculated into LB medium supplemented with kanamycin and cultured overnight at 37°C. Further LB medium was added for preculture, and IPTG was added to a final concentration of 0.5 mM to induce expression overnight. The following day, pellets containing each enzyme were collected and frozen.

[0165] The frozen pellet was suspended in equilibration buffer (20 mM Tris-HCl (pH 8.4), 250 mM NaCl, 20 mM imidazole), and lysozyme and TCEP were added and further suspended. Triton X-100 was added and the suspension was then subjected to ultrasonic treatment, DNase I was added, the mixture was shaken, and the mixture was centrifuged to collect the supernatant. The obtained sample was applied to a column and purified with elution buffer (20 mM Tris-HCl (pH 8.4), 250 mM NaCl, 20 mM imidazole), and then dialyzed twice against TES buffer (10 mM TES, 150 mM NaCl, pH 7.0). Purification of the target enzyme was confirmed using a microspectrophotometer.

[0166] To clarify the thermal stability of these enzymes when immobilized on a support, a glycated protein sensor was fabricated. A platinum electrode was subjected to silane coupling treatment to introduce amino groups onto the electrode surface. Furthermore, bovine serum albumin (BSA) was added to a TES buffer solution, and the enzymes were added and stirred. A predetermined amount of glutaraldehyde solution was then added to the mixture of enzymes and BSA and stirred. 1 μL of this solution was dropped onto the platinum electrode with the amino groups introduced, and the mixture was thoroughly dried to obtain a sensor.

[0167] 200 μL of HEPES buffer containing 50 μM F-Lys was dropped onto each sensor, and current output values ​​were obtained. Furthermore, power output values ​​for F-Lys were obtained under the following conditions: each sensor was immersed in HEPES buffer heated to 50°C for 20 minutes, and after 20 minutes at 50°C, it was immersed in HEPES buffer heated to 65°C for 20 minutes. The power output value before the temperature treatment was set to 100%, and the ratio of the power output value after the temperature treatment to the power output value before the temperature treatment was calculated as the residual activity. A t-test was used for statistical testing.

[0168] A comparison of the residual activities of Example 6 and Example 2 is shown in Figure 8(A), and a comparison of the residual activities of Example 7 and Example 3 is shown in Figure 8(B). It was shown that Example 6 had a significantly higher residual activity after heat treatment at 65°C when immobilized on a support than Example 2, which is the enzyme before amino acid modification, and Example 7 had a significantly higher residual activity after heat treatment at 50°C to 65°C when immobilized on a support than Example 3, which is the enzyme before amino acid modification.

[0169] Verification of lysine residues involved in the thermostability of the FAOD of Example 1 (1) From the experimental results of the above-mentioned variants, it was confirmed that the lysine residues of Example 1 confer thermostability to the FAOD. Therefore, an experiment was conducted to verify which lysine residues in Example 1 play an important role in the thermostability of the FAOD. Variant 1 (SEQ ID NO: 27) in which the lysines at positions 64, 84, 87, 128, 157, 166, 167, 394, and 397 of Example 1 were substituted with arginine; variant 2 (SEQ ID NO: 28) in which the lysines at positions 112, 137, 152, 247, 254, 255, 267, 277, and 438 of Example 1 were substituted with arginine; and variant 3 (SEQ ID NO: 29) in which the lysines at positions 34, 177, 233, 234, 297, 309, 413, 424, 430, and 443 of Example 1 were substituted with arginine. These variants were each prepared by the same method as described above. Furthermore, glycated protein sensors were prepared by immobilizing each of Example 1 and variants 1 to 3.

[0170] 200 μL of HEPES buffer containing 50 μM F-Lys was added dropwise to each sensor, and the current output value was obtained 120 seconds after the addition of F-Lys. Furthermore, each sensor was immersed in HEPES buffer heated to 50°C for 20 minutes, and then immersed in HEPES buffer heated to 60°C for 20 minutes. The power output value for F-Lys was obtained under the following conditions. The power output value before the temperature treatment was defined as 100%, and the ratio of the power output value after the temperature treatment to the power output value before the temperature treatment was calculated as the residual activity. Statistical testing was performed using the Bonferroni method after one-way ANOVA.

[0171] The residual activity after heating at 50°C is shown in Figure 9(A), and the residual activity after heating at 50°C and then heating at 60°C is shown in Figure 9(B). All variants showed significantly reduced residual activity compared to Example 1, suggesting that the lysine substituted with arginine in variants 1 to 3 plays an important role in the thermal stability of Example 1. Furthermore, when the residual activities of the variants were compared, the residual activity of variant 3 was significantly reduced compared to variants 1 and 2. Therefore, it was shown that the lysine substituted with arginine in variant 3 is particularly important for the thermal stability of Example 1.

[0172] - Study of a method for evaluating the thermal stability of FAOD immobilized on a support In order to evaluate the thermal stability of FAOD immobilized on a support using a simpler method than electrodes, the change in the thermal stability of FAOD immobilized on a support in a liquid phase and in an unimmobilized state was examined. Here, BSA was used as the support. The FAOD used was the FAOD from Examples 1 to 3 and an Example (hereinafter referred to as "Example 8") which is a FAOD consisting of the amino acid sequence shown in SEQ ID NO: 20 and is a variant of Example 3.

[0173] Furthermore, a variant of Example 1 (hereinafter referred to as "Variant 4") was prepared. Variant 4 had the following modifications in the amino acid sequence shown in SEQ ID NO: 1: (1) when the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 1 were aligned, the amino acid sequence shown in SEQ ID NO: 2 was lysine, and at positions other than lysine in the amino acid sequence shown in SEQ ID NO: 1, the non-lysine amino acid was substituted with lysine; and (2) at positions other than lysine in the amino acid sequence shown in SEQ ID NO: 2 and lysine in the amino acid sequence shown in SEQ ID NO: 1, the lysine was substituted with the amino acid at that position in the amino acid sequence shown in SEQ ID NO: 2. The amino acid substitutions in Variant 4 are shown in Table 10, and its amino acid sequence is shown in SEQ ID NO: 30.

[0174]

[0175] A Hepes buffer (10 mM Hepes + 150 mM NaCl, pH 8.0) containing 1 mg / mL FAOD (Examples 1, 2, 8, and Modified Form 3) and BSA was prepared. To immobilize FAOD to BSA, a predetermined amount of glutaraldehyde was further added. After incubation at 25°C for 10 minutes, 90 μL of ice-cold 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. This method yielded an enzyme solution containing FAOD immobilized to BSA by the addition of glutaraldehyde, and an enzyme solution containing FAOD not immobilized to BSA by not adding glutaraldehyde. The enzyme activity was measured after the enzyme solution was kept on ice and after it was heated to 48°C on a heat block and kept for 15 minutes. The activity of the enzyme solution kept on ice was set to 100, and the ratio of the activity obtained after treatment at 48°C was calculated as the residual activity.

[0176] Based on the results of measuring the residual activity when BSA was not immobilized and when BSA was immobilized, the rate of change in residual activity was calculated using the following formula to quantify the effect of immobilization to BSA on the residual activity of FAOD. The results are shown in Table 11.

[0177]

[0178]

[0179] The FAOD of Example 1 had a higher residual activity when immobilized with BSA than other FAODs, and its residual activity when not immobilized with BSA was shown to be significantly improved by immobilization with BSA. On the other hand, the residual activity of the FAODs of Examples 2 and 3 decreased with immobilization with BSA. This was the same trend as the measurement results of the glycated protein measurement sensors using Examples 2 and 3 described above. Therefore, this evaluation method can be used to estimate the thermal stability of FAODs when used in glycated protein measurement sensors.

[0180] Furthermore, the residual activity of variant 4, which is a variant of Example 1, was reduced by immobilization with BSA, whereas the residual activity of variant 8, which is a variant of Example 3, was improved by immobilization with BSA, suggesting that the lysine residue in Example 1 plays an important role in the thermal stability of Example 1 when immobilized on a support. It was also shown that the thermal stability of FAODs other than those of Example 1 when immobilized with BSA can be improved by substituting lysine for the amino acid at the position corresponding to the lysine in Example 1.

[0181] Verification of lysine residues involved in the thermal stability of the FAOD of Example 1 (2) From the above results, it became clear that the lysines at positions 34, 177, 233, 234, 297, 309, 413, 424, 430, and 443 of the FAOD of Example 1 play particularly important roles in the thermal stability upon immobilization with BSA. Therefore, important lysine residues were further narrowed down from among these lysine residues. Variant 5 (SEQ ID NO: 31) in Example 1, in which lysines at positions 233, 234, and 297 were substituted with arginine; variant 6 (SEQ ID NO: 32) in Example 1, in which lysines at positions 309 and 413 were substituted with arginine; variant 7 (SEQ ID NO: 33) in Example 1, in which lysines at positions 424, 430, and 443 were substituted with arginine; and variant 8 (SEQ ID NO: 34) in Example 1, in which lysines at positions 34 and 177 were substituted with arginine, were each prepared by the same method as described above. Furthermore, the residual activity of each FAOD in the liquid phase with and without BSA immobilization was measured by the same method as described above. In this experiment, the concentration of BSA in the Hepes buffer was 2.64% (w / v).

[0182] Table 12 shows the residual activity of each FAOD when not immobilized with BSA, the residual activity when immobilized with BSA, and the rate of change in residual activity. Variants 6 and 7 showed a negative rate of change in residual activity compared to Example 1 and other variants. These results suggest that the lysine substituted with arginine in variants 6 and 7 plays a particularly important role in the thermal stability of Example 1 when immobilized with BSA. Therefore, it is predicted that the thermal stability of a FAOD homologous to Example 1 when immobilized with BSA will be improved if the amino acids corresponding to these positions in Example 1 are substituted with lysine.

[0183]

[0184] Verification of lysine residues involved in the thermal stability of the FAOD of Example 1 (4) Comparative Example 4 [Accession Number: BAD54824] is a known FAOD derived from Aspergillus oryzae, and the amino acid sequence of Comparative Example 4 (SEQ ID NO: 24) has a high homology of 95.1% to the amino acid sequence of Example 1. Meanwhile, in Comparative Example 4, the amino acid corresponding to the lysine at position 247 in Example 1 is arginine, and the amino acid corresponding to the lysine at position 277 in Example 1 is asparagine. Therefore, the effect of the lysines at positions 247 and / or 277 in Example 1 on the thermal stability of Example 1 was analyzed.

[0185] In addition to the FAODs of Example 1 and Comparative Example 4, variant 9 (SEQ ID NO: 35) in which the lysines at positions 247 and 277 of Example 1 were substituted with arginine was prepared by the same method as described above. The residual activity of each FAOD when immobilized in BSA in a liquid phase was measured. Statistical testing was performed using the Bonferroni method after one-way ANOVA. The results are shown in Table 13. The residual activity of Example 1 was significantly higher than that of Comparative Example 4 and variant 9, suggesting that the lysines at positions 247 and / or 277 of Example 1 play an important role in the thermal stability of the FAOD when immobilized.

[0186] Different letters indicate that the statistical test was p<0.001.

[0187] - Improving the thermal stability of FAOD based on the lysine arrangement of Example 1 Comparative Example 5 [Accession Number: AAB88209] is a known FAOD derived from Aspergillus fumigatus, and the amino acid sequence of Comparative Example 5 (SEQ ID NO: 25) has 79.2% homology with the amino acid sequence of Example 1. In order to increase the thermal stability of the FAOD of Comparative Example 5 when immobilized on a support, the FAOD of Example 9 was prepared having the following amino acid substitutions in the amino acid sequence of Comparative Example 5.

[0188] In Example 9, in the amino acid sequence of Comparative Example 5 shown in SEQ ID NO: 25, (1) when the amino acid sequence shown in SEQ ID NO: 25 was aligned with the amino acid sequence shown in SEQ ID NO: 1, at a position which is lysine in the amino acid sequence shown in SEQ ID NO: 1 but is an amino acid other than lysine in the amino acid sequence shown in SEQ ID NO: 25, the non-lysine amino acid was substituted with lysine, and (2) at a position which is an amino acid other than lysine in the amino acid sequence shown in SEQ ID NO: 25 but is lysine in the amino acid sequence shown in SEQ ID NO: 1, a substitution of lysine with the amino acid at that position in the amino acid sequence shown in SEQ ID NO: 25 was introduced. The amino acid substitutions in the FAOD of Example 9 are as shown in Table 14, and its amino acid sequence is shown in SEQ ID NO: 23.

[0189]

[0190] The residual activity of each FAOD in the liquid phase when immobilized on BSA was measured for the FAODs of Example 1, Comparative Example 5, and Example 9. Statistical testing was performed using the Bonferroni method after one-way ANOVA. The results are shown in Table 15. The residual activity of Comparative Example 5 was significantly lower than that of Example 1, but the residual activity of Example 9 was significantly improved compared to Comparative Example 5. Therefore, it was confirmed that the introduction of the above-mentioned amino acid substitutions can increase the thermal stability of FAOD when immobilized on a support.

[0191] Different letters indicate that the statistical test was p<0.001.

[0192] Several embodiments and examples of the present disclosure have been described above, but these embodiments and examples exemplify the present disclosure. For example, the above embodiments have been described in detail to clearly explain the present disclosure, and additional changes in dimensions, configurations, materials, and circuits may be made as necessary. Note that embodiments that combine any one or more features of the present disclosure described above are also included within the scope of the present disclosure. The claims encompass numerous modifications to the embodiments without departing from the technical spirit of the present disclosure. Therefore, the embodiments and examples disclosed herein are provided for illustrative purposes and should not be considered to limit the scope of the present disclosure.

[0193] REFERENCE SIGNS LIST 10 Glycated protein sensor 12 Enzyme layer 14 Hydrogen peroxide detection unit 16 Detection surface 18 Silane coupling agent 20 Glycated protein sensor 22 Fructosyl amino acid oxidase layer 24 Protease layer

Claims

1. A fructosyl amino acid oxidase that satisfies one or more of the following (1) and (2): (1) The amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 1 in which 1 to 15 amino acids have been deleted, substituted, added, and / or inserted. (2) An amino acid sequence having 30% or more homology with the amino acid sequence described in (1) above, which satisfies the following items (2-1) and (2-2): (2-1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid at position 247 in the amino acid sequence shown in SEQ ID NO: 1 is lysine. (2-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid at position 430 and / or 443 in the amino acid sequence shown in SEQ ID NO: 1 is lysine.

2. A fructosyl amino acid oxidase according to claim 1, further satisfying (3) or (4). (3) The residual activity after treatment at 48°C when immobilized on a support is higher than the residual activity after treatment under the same conditions when not immobilized on a support. (4) The residual activity after treatment at 65 to 80°C for 20 minutes while immobilized on a support is 70% or more.

3. The fructosyl amino acid oxidase according to claim 2, wherein the state of being immobilized on a support in (3) and / or (4) is a state of being immobilized on a support by crosslinking with an amine-reactive crosslinking agent.

4. The fructosyl amino acid oxidase of claim 3, which is a recombinant protein.

5. A fructosyl amino acid oxidase as described in claim 4, which has a homology of 74% or more with the amino acid sequence shown in SEQ ID NO:

1.

6. The fructosyl amino acid oxidase according to any one of claims 1 to 5, which has a reactivity specific to fructosyl lysine.

7. 7. The fructosyl amino acid oxidase according to claim 6, wherein the reactivity to fructosyl valine or a peptide containing fructosyl valine is 20 or less, relative to the reactivity to fructosyl lysine taken as 100.

8. 7. The fructosyl amino acid oxidase according to claim 6, wherein the reactivity to fructosyl glycine is 20 or less when the reactivity to fructosyl lysine is taken as 100.

9. A support; and fructosyl amino acid oxidase immobilized on the support, The fructosyl amino acid oxidase is the fructosyl amino acid oxidase according to any one of claims 1 to 5. Glycated protein sensor.

10. The glycated protein sensor according to claim 9 , wherein the fructosyl amino acid oxidase is immobilized on the support by cross-linking with an amine-reactive cross-linker.

11. The glycated protein sensor according to claim 10 , further comprising a hydrogen peroxide detection unit.

12. The glycated protein sensor according to claim 11 , wherein a support layer including the support and the hydrogen peroxide detection unit are laminated.

13. A method for measuring a glycated protein, which detects a glycated protein by a reaction of fructosyl amino acid oxidase immobilized on a support, comprising: The fructosyl amino acid oxidase is the fructosyl amino acid oxidase according to any one of claims 1 to 5. Method for measuring glycated proteins.

14. A method for producing fructosyl amino acid oxidase, comprising: a step of substituting, with lysine, amino acids in an amino acid sequence of a fructosyl amino acid oxidase having 30% or more homology with the amino acid sequence of SEQ ID NO: 1, which amino acids correspond to one or more positions selected from positions 309, 413, 424, 430, and 443 of the amino acid sequence of SEQ ID NO: 1 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 1; the thermostability of the fructosyl amino acid oxidase comprising the amino acid sequence after the amino acid substitution when immobilized on a support is higher than the thermostability of the fructosyl amino acid oxidase comprising the amino acid sequence before the amino acid substitution when immobilized on a support; A method for producing fructosyl amino acid oxidase.

15. A method for producing fructosyl amino acid oxidase, comprising: The method comprises substituting any one of the following amino acids (8-1) to (8-3) with lysine in an amino acid sequence of a fructosyl amino acid oxidase having a homology of 30% or more with the amino acid sequence shown in SEQ ID NO: 1: the thermal stability of the fructosyl amino acid oxidase comprising the amino acid sequence after the amino acid substitution when immobilized on a support is higher than the thermal stability of the fructosyl amino acid oxidase comprising the amino acid sequence before the amino acid substitution when immobilized on a support. (8-1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids in the amino acid sequence correspond to positions 64, 84, 87, 128, 157, 166, 167, 394, and 397 of the amino acid sequence shown in SEQ ID NO:

1. (8-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids in the amino acid sequence correspond to positions 112, 137, 152, 247, 254, 255, 267, 277, and 438 of the amino acid sequence shown in SEQ ID NO:

1. (8-3) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acids in the amino acid sequence correspond to positions 34, 177, 233, 234, 297, 309, 413, 424, 430, and 443 of the amino acid sequence shown in SEQ ID NO:

1.

16. A method for producing fructosyl amino acid oxidase, comprising: The method comprises the steps of: making the following amino acid substitutions (9-1) and (9-2) in an amino acid sequence of a fructosyl amino acid oxidase having a homology of 30% or more with the amino acid sequence shown in SEQ ID NO: 1; the thermal stability of the fructosyl amino acid oxidase comprising the amino acid sequence after the amino acid substitution when immobilized on a support is higher than the thermal stability of the fructosyl amino acid oxidase comprising the amino acid sequence before the amino acid substitution when immobilized on a support. (9-1) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, a position that is a lysine in the amino acid sequence shown in SEQ ID NO: 1 but an amino acid other than lysine in the amino acid sequence is substituted with a lysine. (9-2) When the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, a position that is an amino acid other than lysine in the amino acid sequence shown in SEQ ID NO: 1 but is lysine in the amino acid sequence is substituted with the amino acid at that position in the amino acid sequence shown in SEQ ID NO:

1.

17. The thermal stability is in a state where the polymer is fixed to the support by crosslinking with an amine-reactive crosslinker. A method for producing the fructosyl amino acid oxidase according to any one of claims 14 to 16.

18. the residual activity of the fructosyl amino acid oxidase after treatment at 48°C in a state immobilized on a support is higher than the residual activity after treatment under the same conditions in a state where the fructosyl amino acid oxidase is not immobilized on a support, and / or the residual activity of the fructosyl amino acid oxidase after treatment at 65°C to 80°C for 20 minutes in a state where the fructosyl amino acid oxidase is immobilized on a support is 70% or more; A method for producing the fructosyl amino acid oxidase according to claim 17.

19. The method for producing a fructosyl amino acid oxidase according to claim 18, wherein the homology is 74% or more.

20. A fructosyl amino acid oxidase comprising an amino acid sequence of any one of SEQ ID NOs: 2 to 23, or an amino acid sequence in which 1 to 15 amino acids have been deleted, substituted, added, and / or inserted in said amino acid sequence.