Fructosyl-amino acid oxidase, gene, expression vector, glycated protein sensor, glycated protein measurement method, and fructosyl-amino acid oxidase production method

Abstract

[Problem] Technology for enhancing the measurement accuracy and the stability of glycated protein sensors containing an immobilized enzyme is essential. However, there was no known enzyme capable of enhancing the accuracy or stability of measurements using glycated protein sensors. [Solution] The present invention provides a fructosyl-amino acid oxidase comprising an amino acid sequence in which, when aligned with the amino acid sequence represented by SEQ ID NO: 1, any amino acid corresponding to a position selected from the group consisting of position 5, position 58, position 225, position 277, and position 440 in the amino acid sequence represented by SEQ ID NO: 1 has been substituted.

Description

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

[0001] The present invention relates to fructosyl amino acid oxidase, a gene encoding the amino acid sequence of fructosyl amino acid oxidase, an expression vector containing the gene, a glycated protein sensor comprising fructosyl amino acid oxidase immobilized on a support, a method for measuring glycated proteins, which detects glycated proteins through the reaction of fructosyl amino acid oxidase immobilized on a support, and a method for producing fructosyl amino acid oxidase.

[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, treating glycated amino acids (hereinafter sometimes referred to as "glycated amino acids" or "fructosyl amino acids") among the amino acids or peptides or peptides containing glycated amino acids (hereinafter sometimes referred to as "glycated peptides" or "fructosyl peptides") with fructosyl amino acid oxidase to generate hydrogen peroxide, and in a third step, converting the hydrogen peroxide into a color-developing reaction and measuring the absorbance, or detecting electrons released by the decomposition of hydrogen peroxide at an electrode (see Patent Document 1).

[0004] Many known inventions feature enzymes modified to improve the accuracy of measuring specific glycated amino acids or glycated peptides. Examples include amadoriases that can be used to measure glycated hemoglobin accurately in samples containing glycated abnormal hemoglobin and that are compatible with various genotypes (see Patent Document 2), amadoriases obtained by substituting amino acids in a specific amino acid sequence to improve the specific activity toward glycated substrates (see Patent Document 3), amadoriases obtained by substituting amino acids in a specific amino acid sequence to maintain activity in the presence of surfactants (see Patent Documents 4 and 5), fructosyl amino acid oxidases obtained by substituting amino acids in a specific amino acid sequence to improve thermostability (see Patent Documents 6 and 7), amadoriases that are less susceptible to the effects of oxygen concentration (see Patent Document 8), and a method for measuring fructosyl lysine using fructosyl amino acid oxidase from Aspergillus oryzae strain RIB40 (see Patent Document 9).

[0005] International Publication No. WO 2019 / 221264 International Publication No. WO 2019 / 045052 International Publication No. WO 2016 / 159384 International Publication No. WO 2016 / 072520 International Publication No. WO 2015 / 020200 International Publication No. WO 2015 / 096621 JP 2015-177790 A Publication No. WO 2016 / 063984 JP 2009-000084

[0006] For glycosylated protein sensors equipped with enzymes, technology to improve measurement accuracy and sensor stability is essential. However, no enzymes that can improve measurement accuracy or sensor stability using glycosylated protein sensors were known.

[0007] In other words, the problem to be solved by the present invention is to provide a fructosyl amino acid oxidase that has physicochemical properties, such as high enzymatic activity and high thermal stability, that contribute to the accuracy of measurements using a glycated protein sensor and the stability of the sensor.

[0008] The present inventors discovered a fructosyl amino acid oxidase with novel physicochemical properties from among genes of unknown function, and further discovered that the physicochemical properties of the fructosyl amino acid oxidase can be improved by substituting specific amino acids on the fructosyl amino acid oxidase, thereby arriving at the present invention.

[0009] That is, the present invention relates to a fructosyl amino acid oxidase having an amino acid sequence in which, when aligned with the amino acid sequence shown in SEQ ID NO: 1, any amino acid corresponding to a position selected from the group consisting of positions 5, 58, 59, 98, 225, 277, 285, 355, and 440 in the amino acid sequence shown in SEQ ID NO: 1 has been substituted.

[0010] The present invention may provide a fructosyl amino acid oxidase having higher fructosyl amino acid oxidase activity and / or higher thermostability than a fructosyl amino acid oxidase having no amino acid substitution, such as a fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 1 or 199. More specifically, the fructosyl amino acid oxidase has fructosyl amino acid oxidase activity and / or thermostability that are 4% or more higher than a fructosyl amino acid oxidase having no amino acid substitution, such as a fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 1 or 199.

[0011] Another aspect of the present invention may be a fructosyl amino acid oxidase that has higher activity and / or thermal stability when immobilized on a support than a fructosyl amino acid oxidase in which no amino acid substitutions are present, such as a fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1 or 199.

[0012] Another aspect of the present invention is a fructosyl amino acid oxidase that exhibits higher activity after heat treatment compared to the activity of a fructosyl amino acid oxidase containing no amino acid substitutions after heat treatment, such as a fructosyl amino acid oxidase consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 199. The heat treatment may be carried out at 45°C or 48°C for 15 minutes, for example.

[0013] Another aspect of the present invention is a fructosyl amino acid oxidase that has higher thermostability against heat treatment compared to fructosyl amino acid oxidases without amino acid substitution, such as a fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1 or 199. The heat treatment conditions are, for example, 45°C or 48°C for 15 minutes.

[0014] Another aspect of the present invention may be a fructosyl amino acid oxidase having a reactivity specific to fructosyl lysine, such as a fructosyl amino acid oxidase whose reactivity with fructosyl valine, a peptide containing fructosyl valine, and fructosyl glycine is 20 or less, relative to the reactivity with fructosyl lysine taken as 100.

[0015] Another aspect of the present invention is a fructosyl amino acid oxidase having an amino acid sequence showing 75% or more homology to the amino acid sequence set forth in SEQ ID NO: 1. The amino acid sequence of the fructosyl amino acid oxidase of the present invention may be an amino acid sequence that satisfies at least any of the following (1) to (9): (1) The amino acid corresponding to position 5 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (2) The amino acid corresponding to position 58 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (3) The amino acid corresponding to position 59 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (4) The amino acid corresponding to position 98 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (5) The amino acid corresponding to position 225 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.(6) The amino acid corresponding to position 277 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (7) The amino acid corresponding to position 285 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (8) The amino acid corresponding to position 355 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (9) The amino acid corresponding to position 440 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0016] Another aspect of the present invention is a fructosyl amino acid oxidase having an amino acid sequence that shows 79% or more homology to the amino acid sequence shown in SEQ ID NO: 1, wherein, when aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 58 in the amino acid sequence shown in SEQ ID NO: 1 is any of histidine, serine, threonine, glycine, alanine, valine, leucine, isoleucine, and phenylalanine, or any of phenylalanine, alanine, and isoleucine.

[0017] Another aspect of the present invention is a fructosyl amino acid oxidase having the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 198 and 200 to 218. A further aspect of the present invention may be a fructosyl amino acid oxidase having an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been deleted, substituted, added, and / or inserted from the amino acid sequence. The present invention also provides a gene encoding the amino acid sequence of the above-described fructosyl amino acid oxidase, and an expression vector comprising the gene.

[0018] The present invention further provides a glycated protein sensor comprising a fructosyl amino acid oxidase and a support, wherein the fructosyl amino acid oxidase can be immobilized on the support by crosslinking with an amine-reactive crosslinker.

[0019] In the sensor of the present invention, the output value of the sensor after being left standing at a temperature of 65°C or 80°C for 20 minutes may be 60% or more of the output value of the sensor before being left standing. Furthermore, the output value of the sensor after being left standing at a temperature of 75°C and a relative humidity of 60% for one or two days may be 40%, 50%, 60%, or 70% or more of the output value of the sensor before being left standing.

[0020] The present invention further provides a method for measuring glycated proteins, which detects glycated proteins through the reaction of fructosyl amino acid oxidase, and a method for producing fructosyl amino acid oxidase, which comprises an amino acid substitution step of substituting an amino acid corresponding to a position selected from the group consisting of positions 5, 58, 225, 277, and 440 in the amino acid sequence of SEQ ID NO: 1 when the amino acid sequence of fructosyl amino acid oxidase is aligned with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of fructosyl amino acid oxidase before amino acid substitution used in the production method of the present invention may be an amino acid sequence that shows high homology to the amino acid sequence of SEQ ID NO: 1, for example, 50% or more, 60% or more, 70% or more, 75% or more, or 79% or more.

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

[0022] Furthermore, according to the present invention, it is possible to obtain a fructosyl amino acid oxidase having higher enzymatic activity and thermostability than a wild-type fructosyl amino acid oxidase. The production method of the present invention makes it possible to produce a variant having higher activity and / or thermostability from a known fructosyl amino acid oxidase.

[0023] 1 shows the results of an experiment evaluating the thermal stability of a sensor comprising a fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 1. This figure shows the output values ​​(left) and the rate of change in output values ​​(right) of a sensor comprising a fructosyl amino acid oxidase (wild-type) having the amino acid sequence shown in SEQ ID NO: 1 and a sensor comprising a fructosyl amino acid oxidase (M58F variant) having the amino acid sequence shown in SEQ ID NO: 38. Statistical significance between the two groups was calculated using a t-test. In the figure, "***" indicates a p-value of less than 0.001, "**" indicates a p-value of less than 0.01, and "n.s." indicates that no statistically significant difference was confirmed.

[0024] The fructosyl amino acid oxidase of the present invention has high enzymatic activity and high thermostability. Furthermore, the fructosyl amino acid oxidase of one aspect of the present invention has high thermostability when immobilized on a support. The present invention also provides a gene encoding the amino acid sequence of the fructosyl amino acid oxidase and an expression vector containing the gene. Furthermore, the present invention also provides a glycated protein sensor comprising a support and fructosyl amino acid oxidase immobilized on the support, a method for measuring glycated proteins that detects glycated proteins through the reaction of fructosyl amino acid oxidase immobilized on the support, and a method for producing fructosyl amino acid oxidase. The present invention will be described in detail below based on embodiments.

[0025] Fructosyl amino acid oxidase is an enzyme that acts on amino acids 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.

[0026] 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").

[0027] 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").

[0028] The fructosyl amino acid oxidase of the present invention has the following physicochemical properties: it has 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 buffer solutions. Specific examples of buffer solutions include Tris hydrochloride buffer and phosphate buffered saline (PBS).

[0029] The fructosyl amino acid oxidase of the present invention may 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The present inventors have discovered novel fructosyl amino acid oxidases from genes of unknown function, and have identified a fructosyl amino acid oxidase that exhibits significantly high thermostability both in the immobilized and / or unimmobilized state. Based on the amino acid sequence of this fructosyl amino acid oxidase, they have created a modified fructosyl amino acid oxidase.

[0034] The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of a fructosyl amino acid oxidase derived from Aspergillus pseudotamarii of the genus Aspergillus. Although it was previously predicted to be a type of FAD-dependent oxidoreductase based on sequence homology [Accession Number: XP_031920077], it was not known that a protein comprising the amino acid sequence shown in SEQ ID NO: 1 could function as a fructosyl amino acid oxidase. The present inventors have discovered that a fructosyl amino acid oxidase comprising the amino acid sequence shown in SEQ ID NO: 1 has significantly high thermal stability when immobilized on a support and extremely high substrate specificity for specific glycated amino acids and / or glycated peptides.

[0035] Furthermore, the present invention provides a fructosyl amino acid oxidase comprising an amino acid sequence highly homologous to SEQ ID NO: 1, wherein, when aligned with the amino acid sequence shown in SEQ ID NO: 1, one or more amino acids corresponding to positions selected from the group consisting of positions 5, 58, 225, 277, and 440 in the amino acid sequence shown in SEQ ID NO: 1 have been substituted. On the other hand, fructosyl amino acid oxidases consisting of known amino acid sequences are excluded from the scope of the fructosyl amino acid oxidases of the present invention, even if they satisfy the above-mentioned conditions.

[0036] An amino acid sequence that is highly homologous to SEQ ID NO: 1 is an amino acid sequence that is 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more homologous to the amino acid sequence of SEQ ID NO: 1. or 99% or more homologous to the amino acid sequence of the present invention. These homology values ​​are rounded to one decimal place (the same applies hereinafter). Amino acid sequence homology can be calculated, for example, using the default parameters of BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) of the National Center for Biotechnology Information (NCBI).

[0037] The amino acid at position 5 in the amino acid sequence shown in SEQ ID NO: 1 is asparagine. In one embodiment of the amino acid sequence of a fructosyl amino acid oxidase of the present invention, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 5 in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0038] When the fructosyl amino acid oxidase shown in SEQ ID NO: 199 is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 5 in SEQ ID NO: 1 is isoleucine, but the amino acid sequence shown in SEQ ID NO: 199 is excluded from the amino acid sequence of the fructosyl amino acid oxidase of the present invention. Furthermore, when the amino acid sequence of the fructosyl amino acid oxidase shown in GenBank Accession No. BAD54824 and the amino acid sequence of the fructosyl amino acid oxidase disclosed in Patent Document 9 is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 5 in SEQ ID NO: 1 is lysine, but the amino acid sequences of these known fructosyl amino acid oxidases are excluded from the amino acid sequence of the fructosyl amino acid oxidase of the present invention.

[0039] The amino acid at position 58 in the amino acid sequence shown in SEQ ID NO: 1 is methionine. In one embodiment of the amino acid sequence of a fructosyl amino acid oxidase of the present invention, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 58 in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0040] The amino acid at position 59 in the amino acid sequence shown in SEQ ID NO: 1 is glutamic acid. In one embodiment of the amino acid sequence of a fructosyl amino acid oxidase of the present invention, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 59 in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0041] The amino acid at position 98 in the amino acid sequence shown in SEQ ID NO: 1 is glutamic acid. In one embodiment of the amino acid sequence of a fructosyl amino acid oxidase of the present invention, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 98 in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0042] The amino acid at position 225 in the amino acid sequence shown in SEQ ID NO: 1 is glycine. In one embodiment of the amino acid sequence of a fructosyl amino acid oxidase of the present invention, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 225 in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0043] The amino acid at position 277 in the amino acid sequence shown in SEQ ID NO: 1 is lysine. In one embodiment of the amino acid sequence of a fructosyl amino acid oxidase of the present invention, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 277 in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0044] When the amino acid sequence of the fructosyl amino acid oxidase disclosed in Patent Document 9 is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 277 in SEQ ID NO: 1 is asparagine. However, the amino acid sequence of the fructosyl amino acid oxidase disclosed in Patent Document 9 is excluded from the amino acid sequence of the fructosyl amino acid oxidase of the present invention.

[0045] The amino acid at position 285 in the amino acid sequence shown in SEQ ID NO: 1 is glutamic acid. In one embodiment of the amino acid sequence of a fructosyl amino acid oxidase of the present invention, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 285 in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0046] The amino acid at position 355 in the amino acid sequence shown in SEQ ID NO: 1 is aspartic acid. In one embodiment of the amino acid sequence of a fructosyl amino acid oxidase of the present invention, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 355 in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0047] The amino acid at position 440 in the amino acid sequence shown in SEQ ID NO: 1 is glycine. In one embodiment of the amino acid sequence of a fructosyl amino acid oxidase of the present invention, when the amino acid sequence is aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 440 in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0048] Another embodiment of the present invention is a fructosyl amino acid oxidase having an amino acid sequence containing multiple amino acid substitutions as described above. Specifically, the present invention includes a fructosyl amino acid oxidase having an amino acid sequence containing substitutions of amino acids corresponding to 2, 3, 4, 5, 6, 7, 8, or 9 positions selected from the group consisting of positions 5, 58, 59, 98, 225, 277, 285, 355, and 440.

[0049] An example of the amino acid sequence of the fructosyl amino acid oxidase of the present invention is shown below. One aspect of the present invention may be a fructosyl amino acid oxidase that comprises or consists of any of the following amino acid sequences. In another aspect, the present invention may be a fructosyl amino acid oxidase that consists of any of the following amino acid sequences.

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] Another example of the amino acid sequence of the fructosyl amino acid oxidase of the present invention is shown below: In the amino acid sequence shown below, the methionine at position 58 of the amino acid sequence shown in SEQ ID NO: 199 is substituted with any amino acid selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0062] Here, the amino acid sequence shown in SEQ ID NO: 199 is a fructosyl amino acid oxidase derived from Aspergillus fumigatus [Accession Number: AAB88209], and is also referred to as "amadoriase I." The amino acid sequence shown in SEQ ID NO: 199 was found to have 79.2% amino acid identity with the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequences shown in SEQ ID NOs: 200 to 218, obtained by substituting one amino acid in the amino acid sequence shown in SEQ ID NO: 199, were found to have 79.0% amino acid identity with the amino acid sequence shown in SEQ ID NO: 1.

[0063]

[0064] Another embodiment of the present invention is a fructosyl amino acid oxidase having an amino acid sequence set forth in any of the above SEQ ID NOs, in which one or several amino acids have been modified or mutated, or deleted, substituted, added, and / or inserted, and which has physicochemical properties such as high thermostability and high substrate specificity for specific glycated amino acids or glycated peptides, where "one or several amino acids" refers to 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 amino acids.

[0065] The present invention also provides a gene comprising a nucleotide sequence encoding the above-mentioned amino acid sequence. Here, the gene may be DNA or RNA, or may be complementary DNA (cDNA). The present invention also provides an expression vector comprising a gene comprising a nucleotide sequence encoding the following amino acid sequence:

[0066] The fructosyl amino acid oxidase of the present invention may be a recombinant protein obtained by introducing a nucleic acid comprising a nucleotide sequence encoding the fructosyl amino acid oxidase of the present invention into a host such as Escherichia coli, yeast, mammalian cells, insect cells, etc. The recombinant protein may also be a recombinant protein synthesized in a cell-free protein synthesis system.

[0067] One embodiment of the present invention is a fructosyl amino acid oxidase comprising the amino acid sequence described above. In this embodiment, an amino acid sequence that functions as a tag may be added to the N-terminus and / or C-terminus to facilitate purification and detection of the enzyme. Examples of tags include His tag, Myc tag, HA tag, and FLAG tag.

[0068] The fructosyl amino acid oxidase of the present invention has high activity toward a substrate, both when immobilized on a support and / or when not immobilized on a support. In one embodiment of the present invention, the fructosyl amino acid oxidase obtained by the above-mentioned amino acid substitution has higher activity toward a substrate than a fructosyl amino acid oxidase consisting of an amino acid sequence without amino acid substitution. The fructosyl amino acid oxidase of the present invention may have activity improved by 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to the fructosyl amino acid oxidase before amino acid substitution. These values ​​are rounded to one decimal place.

[0069] Furthermore, the activity improvement rate of the fructosyl amino acid oxidase of the present invention compared to the wild-type can be calculated, for example, by the following formula: The calculated activity improvement rate will be a positive value if the activity is improved by the amino acid substitution, and a negative value if the activity is decreased by the amino acid substitution.

[0070]

[0071] Another fructosyl amino acid oxidase of the present invention is a fructosyl amino acid oxidase whose activity, measured after heat treatment of the fructosyl amino acid oxidase immobilized on a support and / or not immobilized on a support, is improved compared to that of the wild-type fructosyl amino acid oxidase. The activity of the fructosyl amino acid oxidase of the present invention after heat treatment may be improved by 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to the fructosyl amino acid oxidase before amino acid substitution. These values ​​are rounded to one decimal place (the same applies hereinafter).

[0072] The fructosyl amino acid oxidase of the present invention is heat stable both when immobilized on a support and / or when not immobilized on a support. In one embodiment of the present invention, the fructosyl amino acid oxidase obtained by the above-described amino acid substitution has higher thermostability than a fructosyl amino acid oxidase consisting of an amino acid sequence without amino acid substitution. Specifically, the fructosyl amino acid oxidase of the present invention may have thermostability improved by 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more compared to the fructosyl amino acid oxidase before amino acid substitution. These values ​​are rounded to one decimal place (the same applies hereinafter).

[0073] A fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 1 is heat-stable when immobilized on a support. A fructosyl amino acid oxidase that is heat-stable when immobilized on a support is 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.

[0074] More specifically, the treatment temperature may be 45°C, 48°C, 50°C, 55°C, 60°C, or 65°C. The treatment time may be 10 to 30 minutes, preferably 15 to 25 minutes, and most preferably 20 minutes. The residual activity after heat treatment may be 30%, 40%, 50%, 60%, or 70% or more.

[0075] More specifically, the fructosyl amino acid oxidase of one embodiment of the present invention is included in a glycated protein sensor. One example of a glycated protein sensor includes fructosyl amino acid oxidase immobilized on a support. The fructosyl amino acid oxidase included in the glycated protein sensor has extremely high residual activity after heat treatment of the protein sensor, so that the output value of the sensor remains high even when placed in a high-temperature environment. Specifically, in the sensor of this embodiment, the output value of the sensor after standing at a temperature of 65°C or 80°C for 20 minutes can be 60% or more of the output value of the sensor before standing. Furthermore, the output value of the sensor after standing at a temperature of 75°C and a relative humidity of 60% for 1 day, 2 days, or 3 days can be 40%, 50%, 60%, or 70% or more of the output value of the sensor before standing.

[0076] 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 45°C, 48°C, 50°C, 55°C, 60°C, or 65°C. The treatment time is 10 to 30 minutes, preferably 15 to 25 minutes, and most preferably 20 minutes.

[0077] 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.

[0078] The fructosyl amino acid oxidase of the present invention obtained by substituting amino acids in the amino acid sequence shown in SEQ ID NO: 1 has improved thermostability compared to the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1. As shown in the Examples, the fructosyl amino acid oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1 has extremely high thermostability compared to other FAODs. Therefore, even if the improvement in thermostability due to amino acid substitution is only a few percent, the effect can be said to be significant.

[0079] Here, the thermostability specifically refers to the thermostability with respect to heat treatment at 45° C. or 48° C. for 15 minutes. The thermostability of fructosyl amino acid oxidase can be evaluated by any method, and one example thereof is a method in which the activity of a heat-treated FAOD and the activity of a non-heat-treated FAOD are measured, and the percentage of the enzyme activity remaining after the heat treatment is calculated as the residual activity.

[0080] Furthermore, when comparing the thermostabilities of two FAODs, the residual activities of each can be compared. For example, the rate of improvement in thermostability due to amino acid substitution can be calculated using the following formula. The calculated rate of improvement in thermostability will be a positive value if the amino acid substitution improves thermostability, and a negative value if the amino acid substitution reduces thermostability.

[0081]

[0082] In one embodiment of the present invention, the fructosyl amino acid oxidase is heat stable when immobilized on a support. Examples of methods for immobilizing an enzyme such as a FAOD on a support include covalent bonding, physical adsorption, ionic bonding, cross-linking, entrapment, and biochemical specific binding. Depending on the enzyme used, an immobilization method may be selected that does not inactivate the enzyme, or multiple immobilization methods may be used in combination.

[0083] In some embodiments, the FAOD, either alone or bound to a carrier, is immobilized on a support by crosslinking with a crosslinking agent. In such cases, the support is a protein separate from the FAOD, and the FAOD is immobilized on the support by mixing the FAOD and the support and then using a crosslinking agent such as glutaraldehyde or an isocyanate derivative. Specific examples of proteins include albumins such as bovine serum albumin (BSA), collagen, and gelatin.

[0084] 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.

[0085] The amine-reactive crosslinking agent reacts with amino groups present on the surface of the fructosyl amino acid oxidase used in the present invention to form crosslinks, and therefore the fructosyl amino acid oxidase becomes stable to heat while immobilized on a support.

[0086] In other embodiments, the support may be a synthetic polymer, a resin, an inorganic material, a polysaccharide, a mineral, a clay, or the like. Specific means for fixing FAODs to a support include fixing FAODs to a support made of fluororesin, ion exchange resin, polyvinyl alcohol resin, hydraulic resin, photocurable resin, solid polymer electrolyte, polyion complex, urethane, etc.; fixing FAODs to these supports by physical interaction between the support and FAODs when charcoal, bone charcoal, silica gel, glass, zeolite, celite, alumina, titanium oxide, ceramic, hydroxyapatite, etc. are used as supports; fixing FAODs to these supports by physical interaction between the support and FAODs when semipermeable membranes such as nylon, cellophane, ethyl cellulose, acetyl cellulose, polystyrene, etc., or phospholipid membranes are used as supports; fixing FAODs to the support by wrapping them in these membranes when polyacrylamide gel, agar, gelatin, carrageenan, sodium alginate gel, calcium alginate gel, chitosan gel, etc. are used as supports.

[0087] The support may also be a carbon material and / or beads. The carbon material may be carbon nanotubes, fullerenes, graphene, etc. The beads may be carbon fine 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 FAODs may be immobilized by being crosslinked to the beads.

[0088] 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.

[0089] The fructosyl amino acid oxidase contained in the sensor of the present invention is heat-stable when immobilized on a support, and is therefore characterized by the ability to obtain highly reliable measurement results over a long period of time. When the sensor of the present invention was left standing at 65°C or 80°C for 20 minutes, it maintained an output value of 70% or more of its pre-standing value. Furthermore, when the sensor of the present invention was left standing at 75°C and 60% relative humidity for one day, it maintained 48% of its pre-standing output value, and when the sensor was left standing for two days, it maintained 29% of its pre-standing output value.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] Furthermore, the present invention provides a method for measuring glycated proteins. The method for measuring glycated proteins of the present invention is a method for measuring glycated proteins, in which glycated proteins are detected by the reaction of fructosyl amino acid oxidase immobilized on a support. The measurement method of the present invention may be a non-diagnostic and / or diagnostic measurement method.

[0101] The present invention further provides a method for producing a fructosyl amino acid oxidase, comprising an amino acid substitution step of substituting an amino acid at a position selected from the group consisting of positions 5, 58, 225, 277, and 440 in the amino acid sequence of SEQ ID NO: 1 when the amino acid sequence of the fructosyl amino acid oxidase is aligned with the amino acid sequence of SEQ ID NO: 1. The activity and / or thermostability of the fructosyl amino acid oxidase produced by this production method, when immobilized on a support and / or when not immobilized on a support, is higher than the activity and / or thermostability of a fructosyl amino acid oxidase in which the amino acid is not substituted.

[0102] The amino acid sequence of the fructosyl amino acid oxidase used herein is one that is highly homologous to the amino acid sequence shown in SEQ ID NO: 1. Specifically, an amino acid sequence that is highly homologous to SEQ ID NO: 1 includes an amino acid sequence that is 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 1 The amino acid sequence may have a homology of 1% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. These homology values ​​are rounded to one decimal place (the same applies hereinafter). Amino acid sequence homology can be calculated, for example, using the default parameters of BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) of the National Center for Biotechnology Information (NCBI).

[0103] The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0104] 1. Analysis of the thermostability and substrate specificity of fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 1

[0105] The thermal stability of fructosyl amino acid oxidase containing the amino acid sequence shown in SEQ ID NO: 1 and a His tag attached to the C-terminus (hereinafter referred to as "wild-type") and FAOD-E (manufactured by Kikkoman Corporation; hereinafter referred to as "comparative example") when immobilized on a support was examined.

[0106] A glycated protein sensor was fabricated using wild-type or comparative enzymes. 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 each enzyme 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 a sensor.

[0107] 200 μL of HEPES buffer containing 50 μM F-Lys was dropped onto each sensor, and a current output value was 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. The output value for F-Lys was obtained in the same manner as in the measurement before the temperature treatment. The output value before the temperature treatment was set to 100%, and the ratio of the output value after the temperature treatment to the output value before the temperature treatment was calculated as the rate of change in the output value.

[0108] The results of measuring the thermal stability of the immobilized enzyme are shown in Figure 1. The sensor equipped with wild-type fructosyl amino acid oxidase (SEQ ID NO: 1) immobilized on a support maintained an output value of 70% or more for F-Lys even after standing at 65 to 80°C for 20 minutes, demonstrating superiority over the comparative example.

[0109] The substrate specificity of the wild-type and comparative samples was also investigated in the liquid phase. The substrates used were fructosyl lysine (F-Lys), fructosyl valyl histidine (F-Val-His), fructosyl valine (F-Val), and fructosyl glycine (F-Gly). At room temperature, 20 μL of substrate solution containing 1 mM substrate was added to each well of a microplate. Then, 10 μL of 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.

[0110] Since it was confirmed that the wild-type and comparative examples had fructosyl amino acid oxidase activity when F-Lys was used as a substrate, the activity toward F-Val-His, F-Val, and F-Gly was measured under the same conditions as for F-Lys. The specific activities of the wild-type and comparative examples toward F-Lys, F-Val-His, F-Val, and F-Gly are shown in the table below. The reactivities of the wild-type fructosyl amino acid oxidase (SEQ ID NO: 1) toward F-Val-His, F-Val, and F-Gly were all less than 1, assuming the reactivity toward F-Lys to be 100. It was confirmed that the wild-type fructosyl amino acid oxidase has F-Lys-specific reactivity.

[0111]

[0112] 2. Analysis of activity and thermostability of the fructosyl amino acid oxidase of the example when not immobilized on a support

[0113] 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 described herein and designed to add a histidine tag to the N-terminus of each inserted protein was introduced into an expression vector having a lactose operon. The expression vector was used to transform Escherichia coli BL21(DE3) Derived strain (manufactured by Nippon Gene Co., Ltd.).

[0114] Transformed E. coli cells were cultured for 2 hours with shaking in LB liquid medium containing 50 μg / mL kanamycin (Nacalai Tesque). These E. coli cells were then cultured at 16°C for 20 hours in LB liquid medium supplemented with isopropyl β-D-thiogalactopyranoside (hereinafter referred to as "IPTG") to a final concentration of 0.1 mM. The collected 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 supernatant of the cell lysate was filtered through a 0.45 μm pore size filter and then applied to EconoFit Nuvia IMAC Columns, Ni-charged (BIO-RAD). The column was washed with 7 column volumes of buffer A and then eluted with elution buffer (20 mM Tris-HCl (pH 7.5) containing 0.25 M sodium chloride and 0.5 M imidazole).

[0115] The resulting eluate was dialyzed against 10 mM sodium phosphate buffer (pH 8.4) containing 150 mM sodium chloride and 50% by volume of glycerol to obtain a sample. The protein concentration in the sample was measured using absorbance at 280 nm.

[0116] The activity and thermal stability of FAODs obtained by substituting amino acids in the amino acid sequence shown in SEQ ID NO: 1 (hereinafter referred to as "Examples") and FAODs containing the amino acid sequence shown in SEQ ID NO: 1 (hereinafter referred to as "wild-type") were evaluated. 50 μL of HEPES buffer containing 0.01 to 0.1 mg / mL of each enzyme (hereinafter referred to as "enzyme dilutions") was dispensed into tubes and maintained on ice or at 45°C on a heat block for 15 minutes. Next, 0.5 wt% TOOS, POD / 4-AA solution (diluted with HEPES buffer (pH 8.0, 0.15 M NaCl) to a concentration of 4.76 μg / mL peroxidase and 0.1 mg / mL 4-aminoantipyrine), and the enzyme dilutions maintained on ice or heat-treated on a heat block were added to wells of a microtiter plate containing 1 mM F-Lys at room temperature and mixed by pipetting. Using a microtiter plate reader set at 37°C, absorbance at 555 nm was measured every 80 seconds for 20 minutes after addition, and the change in absorbance per unit time was calculated as FAOD activity. Hereinafter, the activity measured using the enzyme after keeping it on ice is referred to as "FAOD activity before heat treatment," and the activity measured using the enzyme after heat treatment in a heat block is referred to as "FAOD activity after heat treatment."

[0117] Using the average values ​​of the FAOD activities of the examples and the wild type, the activity improvement rate before and after heat treatment and the thermostability improvement rate were calculated using the following formulas.

[0118]

[0119]

[0120]

[0121] The activity improvement rates and thermostability improvement rates of the enzymes of the examples before and after heat treatment are shown below. Compared to the wild-type enzyme, the enzymes of the examples were confirmed to have at least one of the effects of improved activity before heat treatment, improved activity after heat treatment, and improved thermostability.

[0122] In the table, "-" indicates that the enzyme activity after heat treatment was not confirmed and therefore the value could not be calculated.

[0123] Furthermore, based on the above results, examples with multiple amino acid substitutions were prepared and their enzyme activity and thermal stability were evaluated. The prepared examples are mutants with two amino acid substitutions, N5L and G225N, relative to the amino acid sequence shown in SEQ ID NO: 1. The evaluation method was as described above. As shown below, significant improvements in activity and thermal stability after heat treatment were confirmed compared to mutants with a single amino acid substitution, N5L or G225N.

[0124]

[0125] In addition, the activity and thermal stability of another example were evaluated when not immobilized on a support. The evaluation method was as described above, and heat treatment was performed at 45°C. The activity improvement rate of the enzyme of the example after heat treatment is shown below. It was confirmed that the activity of the enzyme of the example after heat treatment was improved compared to the wild-type enzyme.

[0126]

[0127] Furthermore, another FAOD variant was prepared to confirm whether its activity and thermostability were improved. The other FAOD used was a FAOD containing the amino acid sequence shown in SEQ ID NO: 199. This FAOD is a fructosyl amino acid oxidase derived from Aspergillus fumigatus [Accession Number: AAB88209] and is also called amadoriase I. The amino acid sequence of this FAOD was found to share 79.2% amino acid identity with the amino acid sequence shown in SEQ ID NO: 1.

[0128] The present FAOD and its variants were prepared using the same method as described above, and the activity improvement rate before heat treatment, the activity improvement rate after heat treatment, and the thermostability improvement rate were evaluated. As shown in Table 17, the variants obtained by replacing methionine at position 58 with another amino acid were confirmed to have at least one of the effects of improving activity before heat treatment, improving activity after heat treatment, and improving thermostability compared to the wild-type enzyme.

[0129]

[0130] 3. Analysis of Activity and Thermal Stability of the Fructosyl Amino Acid Oxidase of the Example Immobilized on a Support The activity and thermal stability of the fructosyl amino acid oxidase of the Example and wild-type fructosyl amino acid oxidase immobilized on a support were evaluated. Bovine serum albumin (BSA) was used as the support. A Hepes buffer (10 mM Hepes + 150 mM NaCl, pH 8.0) containing 0.1 mg / mL of enzyme and 0.264% (w / v) BSA was prepared, and glutaraldehyde was added to a concentration of 0.1% (w / v). After incubation at 25°C for 20 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, which contains a primary amine. The enzyme activity was measured after maintaining the mixture on ice at 15°C or on a heat block at 45°C for 15 minutes. The average FAOD activity of each enzyme was used to calculate the activity improvement rate before and after heat treatment, as well as the thermal stability improvement rate.

[0131] The activity improvement rates and thermostability improvement rates of the enzymes of the examples before and after heat treatment are shown below. Compared to the wild-type enzyme, the enzymes of the examples immobilized on a support were confirmed to have at least one of the following effects: improved activity before heat treatment, improved activity after heat treatment, and improved thermostability.

[0132]

[0133] Furthermore, the activity and thermostability of another example enzyme immobilized on a support were evaluated. The evaluation method was as described above, and heat treatment was performed at 45°C. The activity improvement rate and thermostability improvement rate of the enzyme of the example enzyme before and after heat treatment are shown below. Compared to the wild-type enzyme, the enzyme of the example enzyme immobilized on a support was confirmed to have at least one of the following effects: improved activity before heat treatment, improved activity after heat treatment, and improved thermostability.

[0134]

[0135] In addition to the examples and wild-type enzymes, a comparative example (FAOD-E, manufactured by Kikkoman Corporation) was used to evaluate the activity and thermal stability of enzymes immobilized on a support. Bovine serum albumin (BSA) was used as the support, and activity was measured using the method described above. The heat treatment temperature was 45°C. The residual activity rate and thermal stability improvement rate after heat treatment were calculated using the following formulas.

[0136]

[0137]

[0138] The table below shows the residual activity rates of the comparative examples and the wild type, and the residual activity rates and thermal stability improvement rates of the examples. In all examples, a significant improvement in thermal stability was confirmed compared to the wild type.

[0139]

[0140] 4. Evaluation of Sensors Comprising Wild-Type and Modified Fructosyl Amino Acid Oxidases Glycated protein sensors containing wild-type and modified FAODs were prepared and their thermal stability was evaluated. The wild-type FAOD had the amino acid sequence set forth in SEQ ID NO: 1, while the modified FAOD had the amino acid substitution M58F in the wild-type FAOD, i.e., the amino acid sequence set forth in SEQ ID NO: 38. Amino groups were introduced onto the surface of a platinum electrode by silane coupling treatment. The FAOD was then added to a TES buffer solution containing bovine serum albumin (BSA), stirred, and then centrifuged to remove impurities, yielding an enzyme solution. A predetermined amount of glutaraldehyde solution was added to the enzyme solution and rapidly stirred. 1 μL of this solution was dropped onto an amino-introduced platinum electrode and thoroughly dried to obtain a sensor comprising the FAOD and the BSA support. Four sensors were prepared for each of the wild-type FAOD and the variant FAOD, resulting in a total of eight sensors.

[0141] 200 μL of HEPES buffer containing 50 μM F-Lys was dropped onto each sensor, and the resulting change in current was recorded as the sensor's output value. Each sensor used was washed with HEPES buffer, then the moisture was removed with a blower, and the sensor was left to stand at a temperature of 75°C and a relative humidity of 60%. After a certain period of time, the sensor was removed and the sensor output value was recorded in the same manner. This standing and output value recording process was repeated for the same sensor on days 1, 2, 3, 6, and 7, and the output value for each day was recorded. The evaluation results are shown in Figure 2. The sensor equipped with the variant FAOD had significantly higher output values ​​from days 1 to 7 compared to the sensor equipped with the wild-type FAOD, and the rate of change, assuming day 0 as 100%, was also significantly higher from days 1 to 7.

[0142] That is, the sensor of this example is a sensor comprising a support and a FAOD variant fixed to the support, and after leaving the sensor standing for one day under conditions of 75°C and 60% relative humidity, an output value of 73% was maintained, after leaving the sensor standing for two days under the same conditions, an output value of 61% was maintained, after leaving the sensor standing for three days under the same conditions, an output value of 49%, and after leaving the sensor standing for six to seven days under the same conditions, an output value of 27% was maintained.

[0143] 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.

Claims

1. A fructosyl amino acid oxidase having an amino acid sequence in which, when aligned with the amino acid sequence shown in SEQ ID NO:1, any amino acid corresponding to a position selected from the group consisting of 5, 58, 59, 98, 225, 277, 285, 355, and 440 in the amino acid sequence shown in SEQ ID NO:1 has been substituted.

2. The fructosyl amino acid oxidase according to claim 1, which has higher fructosyl amino acid oxidase activity and / or thermostability than a fructosyl amino acid oxidase in which the amino acid is not substituted.

3. The fructosyl amino acid oxidase according to claim 2, which has fructosyl amino acid oxidase activity and / or thermal stability which are 4% or more higher than those of a fructosyl amino acid oxidase in which no amino acid is substituted.

4. The fructosyl amino acid oxidase according to claim 2, wherein the activity and / or thermostability is the activity and / or thermostability when the fructosyl amino acid oxidase is immobilized on a support.

5. The fructosyl amino acid oxidase according to claim 1, wherein the activity of the fructosyl amino acid oxidase after heat treatment is higher than the activity of a fructosyl amino acid oxidase having no amino acid substitution after heat treatment.

6. The fructosyl amino acid oxidase according to claim 5, wherein the heat treatment is carried out at 40°C or 48°C for 15 minutes.

7. The fructosyl amino acid oxidase according to claim 1, which has higher thermostability against heat treatment at 40°C or 48°C for 15 minutes, compared to the fructosyl amino acid oxidase before amino acid substitution.

8. A fructosyl amino acid oxidase according to any one of claims 1 to 7, which has a higher thermostability when immobilized on a support than when not immobilized on a support.

9. The fructosyl amino acid oxidase according to claim 8, which has a specific reactivity to fructosyl lysine.

10. The fructosyl amino acid oxidase according to claim 9, wherein the reactivity to fructosyl lysine is taken as 100 and the reactivity to fructosyl valine, a peptide containing fructosyl valine, and fructosyl glycine is 20 or less.

11. The fructosyl amino acid oxidase according to claim 2, which has an amino acid sequence showing 75% or more homology with the amino acid sequence shown in SEQ ID NO:

1.

12. The fructosyl amino acid oxidase according to claim 11, having an amino acid sequence which satisfies at least any one of the following (1) to (9): (1) the amino acid corresponding to position 5 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, (2) the amino acid corresponding to position 58 of the amino acid sequence shown in SEQ ID NO: 1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (3) The amino acid corresponding to position 59 of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (4) The amino acid corresponding to position 98 of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (5) The amino acid corresponding to position 225 of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.(6) The amino acid corresponding to position 277 of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (7) The amino acid corresponding to position 285 of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (8) The amino acid corresponding to position 355 of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. (9) The amino acid corresponding to position 440 of the amino acid sequence shown in SEQ ID NO:1 is any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

13. A fructosyl amino acid oxidase having an amino acid sequence that is 79% or more identical to the amino acid sequence shown in SEQ ID NO: 1, and when aligned with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 58 in the amino acid sequence shown in SEQ ID NO: 1 is any one of histidine, serine, threonine, glycine, alanine, valine, leucine, isoleucine, and phenylalanine.

14. The fructosyl amino acid oxidase according to claim 13, wherein the amino acid is any one of phenylalanine, alanine, and isoleucine.

15. The fructosyl amino acid oxidase according to claim 2, having an amino acid sequence as set forth in any one of SEQ ID NOs: 2 to 198 and 200 to 218, or an amino acid sequence in which 1 to 5 amino acids have been deleted, substituted, added and / or inserted in said amino acid sequence.

16. A gene encoding the amino acid sequence of the fructosyl amino acid oxidase according to any one of claims 11 to 15.

17. An expression vector comprising the gene according to claim 16.

18. A glycosylated protein sensor comprising the fructosyl amino acid oxidase according to claim 2 and a support on which the fructosyl amino acid oxidase is immobilized.

19. The glycated protein sensor according to claim 18, wherein the fructosyl amino acid oxidase is immobilized on the support by cross-linking with an amine-reactive cross-linker.

20. The sensor described in claim 19, wherein the output value of the sensor after being left standing at 65°C or 80°C for 20 minutes is 60% or more of the output value of the sensor before being left standing, or the output value of the sensor after being left standing at 75°C and a relative humidity of 60% for one day is 40% or more of the output value of the sensor before being left standing.

21. A method for measuring glycated proteins, comprising detecting glycated proteins by the reaction of the fructosyl amino acid oxidase described in claim 2.

22. A method for producing fructosyl amino acid oxidase, comprising an amino acid substitution step of substituting any amino acid corresponding to a position selected from the group consisting of 5, 58, 225, 277, and 440 in the amino acid sequence of SEQ ID NO:1 when the amino acid sequence of fructosyl amino acid oxidase is aligned with the amino acid sequence of SEQ ID NO:

1.

23. The method for producing fructosyl amino acid oxidase according to claim 22, wherein the amino acid sequence of the fructosyl amino acid oxidase has a homology of 79% or more with the amino acid sequence shown in SEQ ID NO:1.

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