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

Abstract

For a glycated protein sensor provided with an immobilized enzyme, a technique for improving the accuracy of measurement and the stability of the glycated protein sensor is essential. However, an enzyme capable of improving the accuracy and stability of measurement using a glycated protein sensor is still unknown. Provided is a fructosyl amino acid oxidase containing an amino acid sequence in which any amino acid corresponding to the 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 is substituted when compared with the amino acid sequence represented by SEQ ID NO: 1.

Description

Technical Field

[0001] This invention relates to fructosyl amino acid oxidase, a gene encoding the amino acid sequence of fructosyl amino acid oxidase, an expression vector containing the gene, a glycated protein sensor having fructosyl amino acid oxidase immobilized on a support, a method for detecting glycated proteins by the reaction of fructosyl amino acid oxidase immobilized on a support, and a method for manufacturing fructosyl amino acid oxidase. Background Technology

[0002] Glycated proteins are measured as indicators for the diagnosis of diabetes and for the management of 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-performance liquid chromatography, immunoassay, and enzymatic methods.

[0003] The enzymatic method for determining glycated proteins is as follows: In the first step, a protease is used to break down the protein into amino acids or peptides; in the second step, fructosyl amino acid oxidase is used to act on the glycated amino acids (hereinafter sometimes referred to as "glycated amino acids" or "fructosyl amino acids") or peptides containing glycated amino acids (hereinafter sometimes referred to as "glycated peptides" or "fructosyl peptides") in these amino acids or peptides to generate hydrogen peroxide; in the third step, the hydrogen peroxide is converted into a colorimetric reaction and the absorbance is measured or the electrons released by decomposing the hydrogen peroxide with an electrode are detected (see Patent Document 1).

[0004] Many inventions are known to be characterized by enzymes modified in ways that improve the accuracy of determination of specific glycated amino acids and glycated peptides. Examples include: amadoric enzymes capable of accurately determining glycated hemoglobin from samples containing abnormal hemoglobin with various genotypes (see Patent Document 2); amadoric enzymes obtained by replacing amino acids in a specific amino acid sequence to improve specific activity against glycated substrates (see Patent Document 3); amadoric enzymes obtained by replacing amino acids in a specific amino acid sequence to leave residual activity in the presence of surfactants (see Patent Documents 4 and 5); fructosyl amino acid oxidases obtained by replacing amino acids in a specific amino acid sequence to exhibit high thermal stability (see Patent Documents 6 and 7); amadoric enzymes that are not easily affected by oxygen concentration (see Patent Document 8); and methods for determining fructosyl lysine using fructosyl amino acid oxidase from Aspergillus oryzae strain RIB40 (see Patent Document 9), etc.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: International Publication No. 2019 / 221264

[0008] Patent Document 2: International Publication No. 2019 / 045052

[0009] Patent Document 3: International Publication No. 2016 / 159384

[0010] Patent Document 4: International Publication No. 2016 / 072520

[0011] Patent Document 5: International Publication No. 2015 / 020200

[0012] Patent Document 6: International Publication No. 2015 / 096621

[0013] Patent Document 7: Japanese Patent Application Publication No. 2015-177790

[0014] Patent Document 8: International Publication No. 2016 / 063984

[0015] Patent Document 9: Japanese Patent Application Publication No. 2009-000084 Summary of the Invention

[0016] The problem that the invention aims to solve

[0017] For glycated protein sensors containing enzymes, techniques to improve measurement accuracy and sensor stability are essential. However, enzymes that can improve the accuracy and stability of measurements using glycated protein sensors are still unknown.

[0018] That is, the problem that this invention aims to solve is to provide a fructose-based amino acid oxidase with physicochemical properties such as high enzyme activity and high thermal stability, which are conducive to the accuracy of measurements and the stability of the sensor when using a glycated protein sensor.

[0019] Methods for solving problems

[0020] The inventors discovered a fructosyl amino acid oxidase with novel physicochemical properties from a gene with unknown function. Furthermore, they found that by replacing specific amino acids on this fructosyl amino acid oxidase, its physicochemical properties could be improved, thus completing this invention.

[0021] That is, the present invention is a fructosyl amino acid oxidase having an amino acid sequence in which any amino acid corresponding to a position in the group consisting of the 5th, 58th, 59th, 98th, 225th, 277th, 285th, 355th and 440th positions of the amino acid sequence shown in Serial No. 1 has been replaced when compared with the amino acid sequence shown in Serial No. 1.

[0022] This invention relates to a fructosyl amino acid oxidase with higher activity and / or thermal stability compared to fructosyl amino acid oxidases without amino acid substitutions, such as fructosyl amino acid oxidases composed of the amino acid sequences shown in Serial No. 1 or 199. More specifically, it refers to a fructosyl amino acid oxidase with at least 4% higher activity and / or thermal stability compared to fructosyl amino acid oxidases without amino acid substitutions, such as fructosyl amino acid oxidases composed of the amino acid sequences shown in Serial No. 1 or 199.

[0023] Another invention may be a fructosyl amino acid oxidase with higher activity and / or thermal stability when immobilized on a support compared to fructosyl amino acid oxidases that have not undergone amino acid substitution, such as fructosyl amino acid oxidases composed of the amino acid sequences shown in serial number 1 or 199.

[0024] Another aspect of the present invention is a fructosyl amino acid oxidase whose activity after heat treatment is higher than that of a fructosyl amino acid oxidase, such as the fructosyl amino acid oxidase consisting of the amino acid sequence shown in Serial No. 1 or 199, which has not undergone amino acid substitution, after heat treatment. The heat treatment conditions are, for example, 45°C or 48°C for 15 minutes.

[0025] Alternatively, another invention may be a fructosyl amino acid oxidase that exhibits higher thermal stability to heat treatment compared to fructosyl amino acid oxidases that have not undergone amino acid substitution, such as fructosyl amino acid oxidases composed of the amino acid sequences shown in Serial No. 1 or 199. The heat treatment conditions described above are, for example, 45°C or 48°C for 15 minutes.

[0026] Another aspect of the present invention may be a fructosyl amino acid oxidase with specific reactivity to fructosyl lysine. For example, the aforementioned reactivity refers to a fructosyl amino acid oxidase whose reactivity to fructosyl lysine is set to 100, and whose reactivity to fructosylvaline, peptides containing fructosylvaline, and fructosylglycine is 20 or less.

[0027] Alternatively, another embodiment of the present invention may be a fructosyl amino acid oxidase having an amino acid sequence that shows more than 75% homology with the amino acid sequence shown in Serial No. 1. The amino acid sequence of the fructosyl amino acid oxidase of the present invention may be an amino acid sequence that at least satisfies any one of (1) to (9) below.

[0028] (1) The amino acid corresponding to the 5th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0029] (2) The amino acid corresponding to the 58th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0030] (3) The amino acid corresponding to the 59th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0031] (4) The amino acid corresponding to the 98th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0032] (5) The amino acid corresponding to the 225th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0033] (6) The amino acid corresponding to the 277th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0034] (7) The amino acid corresponding to the 285th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0035] (8) The amino acid corresponding to the 355th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0036] (9) The amino acid corresponding to the 440th position of the amino acid sequence shown in sequence number 1 is any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0037] Another invention may be a fructosyl amino acid oxidase having an amino acid sequence that shows more than 79% homology with the amino acid sequence shown in Serial No. 1, wherein the amino acid corresponding to position 58 in the amino acid sequence shown in Serial No. 1 when compared with the amino acid sequence shown in Serial No. 1 is any one of histidine, serine, threonine, glycine, alanine, valine, leucine, isoleucine and phenylalanine, or any one of phenylalanine, alanine and isoleucine.

[0038] Another aspect of the present invention is a fructosyl amino acid oxidase having the amino acid sequence shown in any one of serial numbers 2 to 198 and 200 to 218. Furthermore, another aspect of the present invention may be a fructosyl amino acid oxidase having an amino acid sequence formed by deleting, substituting, adding, and / or inserting 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in the aforementioned amino acid sequence. Furthermore, the present invention provides a gene encoding the amino acid sequence of the aforementioned fructosyl amino acid oxidase and an expression vector containing the gene.

[0039] The present invention also provides a glycated protein sensor comprising a fructose-based amino acid oxidase and a support. In the above-described glycated protein sensor, the fructose-based amino acid oxidase can be immobilized on the support by cross-linking with an amine-reactive cross-linking agent.

[0040] The sensor of the present invention can be such that, after being left to stand at a temperature of 65°C or 80°C for 20 minutes, the output value of the sensor is 60% or more of the output value of the sensor before standing. Alternatively, the sensor can be such that, after being left to stand at a temperature of 75°C and a relative humidity of 60% for 1 or 2 days, the output value of the sensor is 40%, 50%, 60%, or 70% or more of the output value of the sensor before standing.

[0041] This invention also provides a method for determining glycated proteins and a method for manufacturing fructosyl amino acid oxidase. The method for determining glycated proteins detects them through a reaction of fructosyl amino acid oxidase. The method for manufacturing fructosyl amino acid oxidase includes the following amino acid substitution step: for the amino acid sequence of the fructosyl amino acid oxidase, any amino acid corresponding to a position selected from the group consisting of positions 5, 58, 225, 277, and 440 in the amino acid sequence shown in Serial No. 1 when the amino acid sequence is compared with that shown in Serial No. 1 is substituted. The amino acid sequence of the fructosyl amino acid oxidase before amino acid substitution used in the manufacturing method of this invention can be an amino acid sequence showing high homology with the amino acid sequence shown in Serial No. 1, for example, it can be 50% or more, 60% or more, 70% or more, 75% or more, or 79% or more.

[0042] Invention Effects

[0043] The fructosyl amino acid oxidase of the present invention is thermally stable in both the immobilized state and / or unimmobilized state. In particular, the thermal stability of the fructosyl amino acid oxidase of the present invention in the immobilized state is advantageous in improving the long-term stability of glycated protein sensors equipped with immobilized enzymes. Because the reduction in enzyme activity during storage is suppressed, highly reliable measurement results can be obtained over a long period of time.

[0044] Furthermore, according to the present invention, a fructosyl amino acid oxidase with higher enzyme activity and thermal stability compared to wild-type fructosyl amino acid oxidase can be obtained. The manufacturing method of the present invention can produce modified forms with high activity and / or thermal stability from known fructosyl amino acid oxidases. Attached Figure Description

[0045] Figure 1 This is a figure showing the results of an experiment evaluating the thermal stability of a sensor containing a fructose-based amino acid oxidase having the amino acid sequence shown in Serial No. 1.

[0046] Figure 2This graph shows the output values ​​(left) and the rate of change of output values ​​(right) of a sensor equipped with the fructose-based amino acid oxidase (wild type) having the amino acid sequence shown in Serial No. 1 and a sensor equipped with the fructose-based amino acid oxidase (modified M58F) having the amino acid sequence shown in Serial No. 38. Statistical significance between the two groups was calculated using a t-test. In the figure, " "Indicates a p-value less than 0.001", "" indicates a p-value less than 0.01, and "ns" indicates that no statistically significant difference was found. Detailed Implementation

[0047] The fructosyl amino acid oxidase of the present invention exhibits high enzymatic activity and high thermostability. Furthermore, the fructosyl amino acid oxidase of one embodiment of the present invention exhibits high thermostability when immobilized on a support. Additionally, the present invention 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 provides a glycated protein sensor comprising a support and a fructosyl amino acid oxidase immobilized on the support, a method for detecting glycated proteins by the reaction of the fructosyl amino acid oxidase immobilized on the support, and a method for manufacturing the fructosyl amino acid oxidase. The present invention will be described in detail below based on embodiments.

[0048] Fructosyl amino acid oxidases are enzymes that generate hydrogen peroxide during the desaccharification of glycosylated amino acids or peptides containing such amino acids by acting on α-amino and / or ε-amino groups. Fructosyl amino acid oxidases (sometimes also referred to as "FAOD") are also known as amadoxins, ketone amine oxidases, or fructosylamine oxidases. Fructosyl amino acid oxidases include fructosyl peptide oxidases (sometimes also referred to as "FPOD" or "FPOX") that act on glycosylated peptides.

[0049] Fructosyl amino acid oxidases use glycosylated amino acids and / or glycosylated peptides as substrates. Specifically, examples of glycosylated amino acids include ε-fructosyllysine (sometimes also called "fructosyl lysine"), α-fructosylvaline (sometimes also called "fructosylvaline"), α-fructosylglycine (sometimes also called "fructosyl glycine"), α-fructosylhistidine (sometimes also called "fructosylhistidine"), α-fructosylleucine (sometimes also called "fructosyl leucine"), and α-fructosylserine (sometimes also called "fructosylserine").

[0050] Specifically, glycosylated peptides can be categorized as peptides consisting of 2 to 10 amino acids, preferably 2 to 6 amino acids, more preferably 2 to 3 amino acids, and containing one or more glycosylated amino acids. Examples include α-fructosylvalinehistidine (sometimes also called "fructosylvalinehistidine").

[0051] The fructose-based amino acid oxidase of the present invention has the following physicochemical properties: it has an optimal pH range of 7 to 9, an effective pH range of 5 to 9, an effective temperature range of 20 to 80°C, and is soluble in buffer solutions. Specific examples of buffer solutions include Tris hydrochloride buffer and phosphate-buffered saline (PBS).

[0052] The fructosyl amino acid oxidase of the present invention can be an enzyme that specifically reacts to a specific glycosylated 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 to fructosyl lysine or a peptide containing fructosyl lysine is set to 100, the reactivity to other glycosylated amino acids or peptides containing other glycosylated amino acids is 20 or less. Preferably, the reactivity to other glycosylated amino acids or peptides containing other glycosylated amino acids is 10 or less, more preferably 5 or less, and even more preferably 1 or less. Other glycosylated amino acids include, for example, one or more selected from fructosylvaline, fructosylglycine, fructosylhistidine, fructosylleucine, and fructosylserine.

[0053] Fructosyl amino acid oxidases are classified into three groups based on substrate specificity. Group 1 fructosyl amino acid oxidases exhibit high specificity for α-amino-glycated amino acids and / or peptides containing such amino acids. Group 2 fructosyl amino acid oxidases exhibit high specificity for ε-amino-glycated amino acids and / or peptides containing such amino acids. Group 3 fructosyl amino acid oxidases exhibit high specificity for both α-amino-glycated amino acids and / or peptides containing such amino acids, as well as ε-amino-glycated amino acids and / or peptides containing such amino acids. The fructosyl amino acid oxidase of the present invention can be a fructosyl amino acid oxidase belonging to any of these groups.

[0054] In another embodiment, the fructosyl amino acid oxidase exhibits high specificity for fructosylvaline-histidine. High specificity for fructosylvaline-histidine means that when the reactivity to fructosylvaline-histidine is set to 100, the reactivity to other glycosylated amino acids or other glycosylated peptides is 20 or less. Preferably, the reactivity to other glycosylated amino acids or other fructosyl peptides is 10 or less, more preferably 5 or less, and even more preferably 1 or less. Other glycosylated amino acids include, for example, one or more selected from fructosylvaline, fructosyllysine, fructosylglycine, fructosylhistidine, fructosylleucine, and fructosylserine. Other fructosyl peptides refer to peptides containing glycosylated amino acids other than fructosylvaline-histidine.

[0055] The fructosyl amino acid oxidase of the present invention can be an enzyme that specifically reacts to a specific glycosylated amino acid or a peptide containing that amino acid. In one embodiment, the fructosyl amino acid oxidase has high specificity for fructosylvaline. High specificity for fructosylvaline means that when the reactivity to fructosylvaline or a peptide containing fructosylvaline is set to 100, the reactivity to other glycosylated amino acids or peptides containing other glycosylated amino acids is 20 or less. Preferably, the reactivity to other glycosylated amino acids or peptides containing other glycosylated amino acids is 10 or less, more preferably 5 or less, and even more preferably 1 or less. Other glycosylated amino acids refer to, for example, one or more selected from fructosyllysine, fructosylglycine, fructosylhistidine, fructosylleucine, and fructosylserine.

[0056] The inventors discovered a novel fructosyl amino acid oxidase from a gene with unknown function. They obtained a fructosyl amino acid oxidase with significantly high thermal stability in both its supported and unsupported states. Therefore, based on the amino acid sequence of this fructosyl amino acid oxidase, they created a modified version of the fructosyl amino acid oxidase.

[0057] The amino acid sequence shown in Serial No. 1 is the amino acid sequence of a fructosyl amino acid oxidase from *Aspergillus pseudotamarii*, a species of *Aspergillus*. Previously, based on sequence homology, it was presumed to be one of the FAD-dependent oxidoreductases [Accession No.: XP_031920077], but it was not known that a protein containing the amino acid sequence shown in Serial No. 1 could function as a fructosyl amino acid oxidase. The inventors have discovered that the fructosyl amino acid oxidase containing the amino acid sequence shown in Serial No. 1 exhibits significantly high thermostability when immobilized on a support and extremely high substrate specificity for specific glycosylated amino acids and / or glycosylated peptides.

[0058] Furthermore, the present invention provides a fructosyl amino acid oxidase containing an amino acid sequence in which one or more amino acids corresponding to positions selected from the group consisting of positions 5, 58, 225, 277, and 440 in the amino acid sequence shown in Serial No. 1 are substituted in an amino acid sequence highly homologous to Serial No. 1. On the other hand, fructosyl amino acid oxidases composed of known amino acid sequences are excluded from the fructosyl amino acid oxidase of the present invention, even if they meet the above conditions.

[0059] Amino acid sequences with high homology to sequence number 1 may contain amino acids with a homology of 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, or 70% or more. Amino acid sequences with homology of 1% or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. These homology values ​​are rounded to one decimal place (the same applies below). Amino acid sequence homology can be calculated, for example, using default parameters in the BLAST database of the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / BLAST / ).

[0060] The amino acid at position 5 of the amino acid sequence shown in Serial No. 1 is asparagine. In one embodiment of the present invention, the amino acid sequence of the fructosyl amino acid oxidase is such that, when compared with the amino acid sequence shown in Serial No. 1, the amino acid corresponding to position 5 of Serial No. 1 is an amino acid sequence selected from any one of the following groups: alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0061] Here, when the fructosyl amino acid oxidase shown in serial number 199 is compared with the amino acid sequence shown in serial number 1, the amino acid corresponding to the 5th position of serial number 1 is isoleucine. However, the amino acid sequence shown in serial number 199 is excluded from the amino acid sequence of the fructosyl amino acid oxidase of the present invention. Furthermore, when the amino acid sequences of the fructosyl amino acid oxidase shown in GenBank accession number BAD54824 and the fructosyl amino acid oxidase disclosed in Patent Document 9 are compared with the amino acid sequence shown in serial number 1, the amino acid corresponding to the 5th position of serial number 1 is lysine. However, 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.

[0062] The amino acid at position 58 of the amino acid sequence shown in Serial No. 1 is methionine. In one embodiment of the present invention, the amino acid sequence of the fructosyl amino acid oxidase is such that, when compared with the amino acid sequence shown in Serial No. 1, the amino acid corresponding to position 58 of Serial No. 1 is an amino acid sequence selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0063] The amino acid at position 59 of the amino acid sequence shown in Serial No. 1 is glutamic acid. In one embodiment of the present invention, the amino acid sequence of the fructosyl amino acid oxidase is such that, when compared with the amino acid sequence shown in Serial No. 1, the amino acid corresponding to position 59 of Serial No. 1 is an amino acid sequence selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0064] The amino acid at position 98 of the amino acid sequence shown in Serial No. 1 is glutamic acid. In one embodiment of the present invention, the amino acid sequence of the fructosyl amino acid oxidase is such that, when compared with the amino acid sequence shown in Serial No. 1, the amino acid corresponding to position 98 of Serial No. 1 is an amino acid sequence selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0065] The amino acid at position 225 of the amino acid sequence shown in Serial No. 1 is glycine. In one embodiment of the present invention, the amino acid sequence of the fructosyl amino acid oxidase is such that, when this amino acid sequence is compared with the amino acid sequence shown in Serial No. 1, the amino acid corresponding to position 225 of Serial No. 1 is an amino acid sequence selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0066] The amino acid at position 277 of the amino acid sequence shown in Serial No. 1 is lysine. In one embodiment of the present invention, the amino acid sequence of the fructosyl amino acid oxidase is such that, when compared with the amino acid sequence shown in Serial No. 1, the amino acid corresponding to position 277 of Serial No. 1 is an amino acid sequence selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0067] Here, when the amino acid sequence of the fructosyl amino acid oxidase disclosed in Patent Document 9 is compared with the amino acid sequence shown in Serial Number 1, the amino acid corresponding to position 277 of Serial Number 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.

[0068] The amino acid at position 285 of the amino acid sequence shown in Serial No. 1 is glutamic acid. In one embodiment of the present invention, the amino acid sequence of the fructosyl amino acid oxidase is such that, when this amino acid sequence is compared with the amino acid sequence shown in Serial No. 1, the amino acid corresponding to position 285 of Serial No. 1 is an amino acid sequence selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0069] The amino acid at position 355 of the amino acid sequence shown in Serial No. 1 is aspartic acid. In one embodiment of the present invention, the amino acid sequence of the fructosyl amino acid oxidase is such that, when this amino acid sequence is compared with the amino acid sequence shown in Serial No. 1, the amino acid corresponding to position 355 of Serial No. 1 is an amino acid sequence selected from any one of the following groups: alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0070] The amino acid at position 440 of the amino acid sequence shown in Serial No. 1 is glycine. In one embodiment of the present invention, the amino acid sequence of the fructosyl amino acid oxidase is such that, when compared with the amino acid sequence shown in Serial No. 1, the amino acid corresponding to position 440 of Serial No. 1 is an amino acid sequence selected from any one of the following groups: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0071] Another embodiment of the present invention is a fructosyl amino acid oxidase having an amino acid sequence containing two or more of the above-described amino acid substitutions. Specifically, examples include fructosyl amino acid oxidases having an amino acid sequence in which the amino acids corresponding to positions 2, 3, 4, 5, 6, 7, 8, or 9 selected from the group consisting of positions 5, 58, 59, 98, 225, 277, 285, 355, and 440 have been substituted.

[0072] The following shows an example of the amino acid sequence of the fructosyl amino acid oxidase of the present invention. One embodiment of the present invention may be a fructosyl amino acid oxidase containing or composed of any of the following amino acid sequences. Another embodiment may be a fructosyl amino acid oxidase composed of any of the following amino acid sequences.

[0073] [Table 1]

[0074]

[0075] [Table 2]

[0076]

[0077] [Table 3]

[0078]

[0079] [Table 4]

[0080]

[0081] [Table 5]

[0082]

[0083] [Table 6]

[0084]

[0085] [Table 7]

[0086]

[0087] [Table 8]

[0088]

[0089] [Table 9]

[0090]

[0091] [Table 10]

[0092]

[0093] [Table 11]

[0094]

[0095] Another example of the amino acid sequence of the fructose-based amino acid oxidase of the present invention is shown. The amino acid sequence shown below is a sequence in which the amino acid at position 58, methionine, of the amino acid sequence shown in sequence number 199 is replaced by any one of the following groups: alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0096] Here, the amino acid sequence shown in sequence number 199 is a fructosyl amino acid oxidase from *Aspergillus fumigatus* [accession number: AAB88209], also known as "Amadoriase I". 79.2% amino acid homology was confirmed between the amino acid sequence shown in sequence number 199 and the amino acid sequence shown in sequence number 1. 79.0% amino acid homology was confirmed between the amino acid sequences shown in sequences 200–218, obtained by substituting one amino acid in the amino acid sequence shown in sequence number 199, and the amino acid sequence shown in sequence number 1.

[0097] [Table 12]

[0098]

[0099] Another embodiment of the present invention is a fructosyl amino acid oxidase having an amino acid sequence formed by modifying, mutating, deleting, substituting, adding, and / or inserting one or more amino acids in the amino acid sequence shown in any of the above sequence numbers, and possessing physicochemical properties such as high thermal stability and high substrate specificity for specific glycosylated amino acids and glycosylated peptides. Here, one or more amino acids refer to 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1 or 2 amino acids.

[0100] Furthermore, the present invention provides a gene containing a base sequence encoding the above-described amino acid sequence. Here, the gene can be DNA or RNA, and can be complementary DNA (cDNA). Additionally, the present invention provides an expression vector containing a gene containing a base sequence encoding the following amino acid sequence.

[0101] The fructosyl amino acid oxidase of the present invention can be a recombinant protein obtained by introducing a nucleic acid containing the base sequence encoding the fructosyl amino acid oxidase of the present invention into a host such as Escherichia coli, yeast, mammalian cells, or insect cells and expressing it. Alternatively, the recombinant protein can also be a recombinant protein synthesized using a cell-free protein synthesis system.

[0102] One embodiment of the present invention is a fructosyl amino acid oxidase containing the above-described amino acid sequence. In this embodiment, an amino acid sequence that functions as a tag can be added to the N-terminus and / or C-terminus to facilitate enzyme purification and detection. Examples of tags include His tags, Myc tags, HA tags, and FLAG tags.

[0103] The fructosyl amino acid oxidase of the present invention exhibits high activity towards substrates in both the state of being immobilized on a support and / or in the state of not being immobilized on a support. In one embodiment of the present invention, the fructosyl amino acid oxidase obtained by the above-described amino acid substitution exhibits higher activity towards substrates compared to the fructosyl amino acid oxidase composed of an amino acid sequence without amino acid substitution. The activity of the fructosyl amino acid oxidase of the present invention can be increased by 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 100% compared to the fructosyl amino acid oxidase before amino acid substitution. These values ​​are obtained by rounding to one decimal place.

[0104] Furthermore, the activity enhancement rate of the fructose-based amino acid oxidase of the present invention compared to the wild type was calculated, for example, using the following mathematical formula. The calculated activity enhancement rate is positive when the activity increases due to amino acid substitution and negative when the activity decreases due to amino acid substitution.

[0105]

[0106] Another fructosyl amino acid oxidase of the present invention is a fructosyl amino acid oxidase whose activity, measured after heat treatment in a state immobilized on a support and / or in a state not immobilized on a support, is increased compared to the wild type. Compared to the fructosyl amino acid oxidase before amino acid replacement, the activity of the heat-treated fructosyl amino acid oxidase of the present invention can be increased by 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 100%. These values ​​are obtained by rounding to one decimal place (the same applies hereinafter).

[0107] The fructosyl amino acid oxidase of the present invention is thermally stable in both the state of being immobilized on a support and / or in the state of not being immobilized on a support. In one embodiment of the present invention, the fructosyl amino acid oxidase obtained by amino acid substitution described above has higher thermal stability compared to the fructosyl amino acid oxidase composed of an amino acid sequence without amino acid substitution. Specifically, the fructosyl amino acid oxidase of the present invention can improve thermal stability by 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more compared to the fructosyl amino acid oxidase before amino acid substitution. These values ​​are obtained by rounding to one decimal place (the same applies hereinafter).

[0108] The fructosyl amino acid oxidase consisting of the amino acid sequence shown in Serial No. 1 is thermally stable when immobilized on a support. A fructosyl amino acid oxidase that is thermally stable when immobilized on a support refers to a fructosyl amino acid oxidase with high residual activity after heat treatment in the immobilized state. For example, a fructosyl amino acid oxidase characterized by a residual activity of over 70% after treatment at 65°C can be cited.

[0109] More specifically, the processing temperature can also be 45°C, 48°C, 50°C, 55°C, 60°C, or 65°C. Furthermore, the processing time can be 10–30 minutes, preferably 15–25 minutes, and most preferably 20 minutes. The residual activity after heat treatment can be 30%, 40%, 50%, 60%, or 70% or more.

[0110] More specifically, as one embodiment of the present invention, a fructosyl amino acid oxidase is included in a glycated protein sensor. An example of a glycated protein sensor is one containing a fructosyl amino acid oxidase immobilized on a support. Because the fructosyl amino acid oxidase contained in this glycated protein sensor has extremely high residual activity after heat treatment of the protein sensor, its output value remains high even when the sensor is placed in a high-temperature environment. Specifically, for the sensor of this embodiment, after standing at 65°C or 80°C for 20 minutes, the sensor's output value can be more than 60% of the sensor's output value before standing. Furthermore, after standing at 75°C and 60% relative humidity for 1 day, 2 days, or 3 days, the output value of the sensor can be more than 40%, 50%, 60%, or 70% of the sensor's output value before standing.

[0111] Another example of a heat-stable fructosyl amino acid oxidase immobilized on a support is a fructosyl amino acid oxidase whose residual activity after heat treatment is higher than that of the fructosyl amino acid oxidase treated under the same conditions without immobilization on a support. More specifically, the treatment temperature is 45°C, 48°C, 50°C, 55°C, 60°C, or 65°C. Furthermore, the treatment time is 10–30 minutes, preferably 15–25 minutes, and most preferably 20 minutes.

[0112] To confirm the thermostability of fructosyl amino acid oxidase, the support immobilized with fructosyl amino acid oxidase can be heated using known methods such as an electric oven or a constant temperature bath, and the residual activity of the fructosyl amino acid oxidase can be measured. Here, residual activity refers to the ratio of enzyme activity after heat treatment at each temperature to enzyme activity before heat treatment.

[0113] The fructosyl amino acid oxidase of the present invention, obtained by substituting amino acids in the amino acid sequence shown in Serial No. 1, exhibits improved thermal stability compared to the fructosyl amino acid oxidase composed of the amino acid sequence shown in Serial No. 1. As shown in the examples, the fructosyl amino acid oxidase composed of the amino acid sequence shown in Serial No. 1 exhibits extremely high thermal stability compared to other FAODs. Therefore, even if the increase in thermal stability due to amino acid substitution is only a few percent, the effect can be considered significant.

[0114] Here, thermal stability specifically refers to the thermal stability after heat treatment at 45°C or 48°C for 15 minutes. The thermal stability of fructosyl amino acid oxidase can be evaluated by any method. As an example, the following method can be listed: measuring the activity of FAOD after heat treatment and the activity of FAOD without heat treatment, and calculating the percentage of enzyme activity remaining after heat treatment as residual activity.

[0115] Furthermore, when comparing the thermal stability of two FAODs, their residual activity can also be compared. For example, the rate of increase in thermal stability due to amino acid replacement can be calculated using the following mathematical formula. The calculated rate of increase in thermal stability is positive when thermal stability is increased by amino acid replacement, and negative when thermal stability is decreased by amino acid replacement.

[0116]

[0117] The fructose-based amino acid oxidase of one embodiment of the present invention is thermally stable when immobilized on a support. Examples of methods for immobilizing enzymes such as FAOD on a support include covalent bonding, physical adsorption, ionic bonding, cross-linking, encapsulation, and specific biochemical binding. An immobilization method that avoids enzyme inactivation can be selected depending on the enzyme used, and two or more immobilization methods can be used in combination.

[0118] In several embodiments, FAOD is fixed to the support, either alone or in a carrier-bound state, by cross-linking with a cross-linking agent. In such cases, the support is a protein different from FAOD, and the FAOD and support are mixed, and the FAOD is fixed to the support using a cross-linking agent such as glutaraldehyde or isocyanate derivatives. Specifically, examples of proteins include albumins such as bovine serum albumin (BSA), collagen, and gelatin.

[0119] Crosslinking agents can be substances capable of intermolecular or intramolecular crosslinking, specifically including glutaraldehyde, isocyanate derivatives, formaldehyde, glyoxal, malondialdehyde, and succinaldehyde. Among these, amine-reactive crosslinking agents are preferred. Amine-reactive crosslinking agents are those that react with the amino groups of proteins to crosslink them; specifically, glutaraldehyde, formaldehyde, N-hydroxy esters, amide esters, and imide esters are examples.

[0120] The amine-reactive crosslinking agent reacts with the amino groups present on the surface of the fructosyl amino acid oxidase used in this invention to crosslink the enzyme, thus making the fructosyl amino acid oxidase thermally stable when immobilized on a support.

[0121] In other embodiments, the support can be made of synthetic polymers, resins, inorganic materials, polysaccharides, minerals, clay, etc. Specifically, methods for fixing FAOD to the support include: fixing FAOD to supports such as fluoropolymers, ion exchange resins, polyvinyl alcohol resins, water-curable resins, photocurable resins, solid polymer electrolytes, polyionomers, and urethanes; fixing FAOD to supports such as charcoal, bone charcoal, silica gel, glass, zeolite, diatomaceous earth, alumina, titanium dioxide, ceramics, and hydroxyapatite through physical interaction between the support and FAOD; fixing FAOD to supports by wrapping FAOD with semi-permeable membranes or phospholipid membranes such as nylon, cellophane, ethyl cellulose, acetyl cellulose, and polystyrene; fixing FAOD to supports by encapsulating FAOD with these membranes when using polyacrylamide gel, agar, gelatin, carrageenan, sodium alginate gel, calcium alginate gel, and chitosan gel; and so on.

[0122] Alternatively, the support can be carbon materials and / or beads. Carbon materials can be carbon nanotubes, fullerenes, graphene, etc. Beads can be carbon microparticles (carbon beads), silica (SiO2) microparticles (silica beads), or beads formed from polysaccharides such as chitin, chitosan, and alginate. Additionally, beads can contain metal microparticles or magnetizable materials, making them magnetic beads. The average particle size of the beads can be above 10 nm or below 200 nm. FAOD can be immobilized by cross-linking with the beads.

[0123] This invention also provides a glycated protein sensor. The glycated protein sensor of this invention comprises at least a support and an enzyme immobilized on the support. The enzyme contains at least a fructosyl amino acid oxidase. The glycated protein sensor of this invention can be a glycated protein sensor for calculating the amount of glycated protein contained in a test sample, i.e., the concentration of glycated protein in the test sample.

[0124] The fructose-based amino acid oxidase contained in the sensor of the present invention is thermally stable when immobilized on a support, thus providing highly reliable measurement results over a long period. The sensor of the present invention retains more than 70% of its original output value when left to stand at 65°C or 80°C for 20 minutes. Furthermore, the sensor retains 48% of its original output value when left to stand at 75°C and 60% relative humidity for 1 day, and retains 29% of its original output value when left to stand at 75°C and 60% relative humidity for 2 days.

[0125] A preferred embodiment of the glycated protein sensor of the present invention includes a support, a fructose-based amino acid oxidase immobilized on the support, and a detection unit. The detection unit detects hydrogen peroxide generated by the fructose-based amino acid oxidase from glycated amino acids and / or glycated peptides. The glycated protein sensor can calculate the amount (concentration) of glycated protein based on the detection result of the detection unit.

[0126] In one embodiment of the glycated protein sensor of the present invention, the detection unit is a hydrogen peroxide detection unit for detecting hydrogen peroxide; specifically, the detection unit can be a hydrogen peroxide electrode. The hydrogen peroxide electrode can detect the electrons released when hydrogen peroxide is decomposed into oxygen in the form of current; therefore, the amount (concentration) of hydrogen peroxide can be calculated based on the detected current value.

[0127] In another embodiment, the detection unit is an optical detection unit that detects hydrogen peroxide by measuring absorbance and light intensity. For example, hydrogen peroxide can be quantified by detecting the colorimetric reaction in the presence of peroxidase and oxidative chromogenic pigments, or based on the luminescence intensity of luminol.

[0128] In another embodiment, the detection unit is an electrochemiluminescence detection unit, using a gold electrode, a platinum electrode, or an indium tin oxide transparent electrode (ITO electrode). Hydrogen peroxide is detected by measuring the luminescence of a luminescent reagent such as luminol. The detection unit can also be a hydrogen peroxide detection unit of other types.

[0129] The test sample for the glycated protein sensor of the present invention can be a solution. The solution can be a bodily fluid, a solution derived from bodily fluids, or a dilution of bodily fluids. The solution may not be a bodily fluid (non-bodily fluid source) solution; it can be a bodily fluid or a mixture of a bodily fluid-derived solution and a non-bodily fluid-derived solution. The solution can be a solution used in sample determination or a solution used in calibration determination. For example, the solution can be a standard solution or a calibration solution. The solution may contain a buffer solution.

[0130] Body fluids can be blood, serum, plasma, lymph, interstitial fluid, intercellular fluid, interstitial fluid, etc., as well as body cavity fluid, serous cavity fluid, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid (medullary fluid), synovial fluid, and aqueous humor. Body fluids can also be digestive fluids such as saliva, gastric juice, bile, pancreatic juice, and intestinal juice, and can include sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, and breast milk. Body fluids can be from animals or humans. Body fluids can be liquids derived from protein-containing foods from animals (e.g., milk, dairy products). Body fluids can also be plant fluids, living plant fluids, or fluids of plant origin. For example, body fluids can be plant juice, honey, or tree sap.

[0131] The solution may contain the substance being measured. For example, the solution may be tears, and the substance being measured may be albumin or glycated albumin contained in tears. Alternatively, the substance being 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.

[0132] The glycated protein sensor of this invention measures glycated proteins. Glycated proteins can be fructosamine. Fructosamine is a general term for glycated proteins contained in blood; specifically, glycated albumin and glycated hemoglobin contained in blood are examples.

[0133] In one embodiment of the present invention, in addition to fructosyl amino acid oxidase, a protease immobilized on a support is also included. Protease refers to a general term for peptide bond hydrolases that hydrolyze and dissimilate proteins and polypeptides. A protease can be an enzyme that breaks down proteins into peptide fragments. When a protein contains glycosylated amino acid residues, the protease can produce one or more of the following groups: glycosylated amino acids, peptide fragments containing glycosylated amino acids, unglycosylated amino acids, and peptide fragments without glycosylated amino acids. Fructosyl amino acid oxidase can react with glycosylated amino acids or peptide fragments containing glycosylated amino acids to produce hydrogen peroxide.

[0134] In one embodiment of the present invention, a support layer such as a thin film layer is formed on a support onto the detection surface of the detection unit, and is disposed thereon. A bonding agent such as a silane coupling agent is used to place the support on or near the detection surface of the detection unit. By forming a bonding layer between the layered support and the detection unit, the support and the detection surface are bonded together.

[0135] Furthermore, the present invention provides a method for determining glycated proteins. This method detects glycated proteins through the reaction of fructosyl amino acid oxidases immobilized on a support. The method of the present invention can be a non-diagnostic and / or diagnostic method.

[0136] Furthermore, the present invention provides a method for manufacturing fructosyl amino acid oxidase. The method for manufacturing fructosyl amino acid oxidase of the present invention includes the following amino acid substitution step: for the amino acid sequence of the fructosyl amino acid oxidase, any amino acid corresponding to a position in the group consisting of positions 5, 58, 225, 277, and 440 in the amino acid sequence shown in Serial No. 1 when the amino acid sequence is compared with that shown in Serial No. 1 is substituted. The fructosyl amino acid oxidase manufactured by this method exhibits higher activity and / or thermal stability in the state of being immobilized on a support and / or not immobilized on a support than the fructosyl amino acid oxidase without amino acid substitution.

[0137] Here, the amino acid sequence of the fructosyl amino acid oxidase uses an amino acid sequence with high homology to the amino acid sequence shown in Serial No. 1. Specifically, the amino acid sequence with high homology to Serial No. 1 may contain 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, or 70% or more of the amino acid sequence of Serial No. 1. The homology values ​​are 71% or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. These homology values ​​are rounded to one decimal place (the same applies below). The homology of amino acid sequences can be calculated, for example, using default parameters in the BLAST database of the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / BLAST / ).

[0138] Example

[0139] The present invention will be further described in detail with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0140] 1. Thermostability and substrate specificity of fructosyl amino acid oxidases with the amino acid sequence shown in Serial No. 1 Sexual analysis

[0141] The thermal stability of fructose-1 amino acid oxidase (hereinafter referred to as "wild type") containing the amino acid shown in sequence number 1 and a His tag added to the C-terminus, as well as FAOD-E (manufactured by Kikkoman Co., Ltd., hereinafter referred to as "comparative example"), in a support-fixed state was verified.

[0142] Glycated protein sensors were fabricated using wild-type or comparative enzymes. A silane coupling treatment was performed on a platinum electrode to introduce amino groups onto the electrode surface. Bovine serum albumin (BSA) was added to a TES buffer, followed by the enzymes and stirring. Then, a predetermined amount of glutaraldehyde solution was added to the enzyme and BSA mixture and stirred. 1 μL of this solution was added dropwise to the amino-introduced platinum electrode, and the electrode was thoroughly dried to obtain the sensor.

[0143] Add 200 μL of HEPES buffer containing 50 μM F-Lys to each sensor to obtain the current output value. Immerse each sensor in HEPES buffer heated to 50℃, 65℃, or 80℃ and let it stand at that temperature for 20 minutes. Then, to eliminate the influence of residual heat, let each sensor stand at 4℃ for 30 minutes. Obtain the F-Lys output value using the same method as the measurement before temperature treatment. Set the output value before temperature treatment as 100%, calculate the ratio of the output value after temperature treatment to the output value before temperature treatment, as the rate of change of the output value.

[0144] The results of the thermostability assay of the immobilized enzyme are shown in... Figure 1 For the sensor equipped with wild-type fructose-based amino acid oxidase (serial number 1) immobilized on the support, it maintained more than 70% of the F-Lys output value after standing at 65℃~80℃ for 20 minutes, which is an advantage over the comparative example.

[0145] In addition, the substrate specificity of wild-type and comparative examples in liquid chromatography was investigated. Fructosyllysine (F-Lys), fructosylvaline-histidine (F-Val-His), fructosylvaline (F-Val), and fructosylglycine (F-Gly) were used as substrates. At room temperature, 20 μL of substrate solution containing 1 mM of the substrate was added to each well of a microplate, along with 10 μL of 0.5 wt% TOOS (manufactured by Dojin Science Research Institute Co., Ltd.), 260 μL of a solution containing 4.76 μg / mL peroxidase and 0.1 mg / mL 4-aminoantipyrine (manufactured by Nakalitesk Co., Ltd.), 5 μL of HEPES buffer (pH 8.0) containing 0.15 M sodium chloride, and an appropriately diluted fructosyl amino acid oxidase solution. The absorbance change at 555 nm was measured at 37 °C. As a negative control, 20 mM potassium phosphate buffer (pH 7.5) was used.

[0146] Both the wild-type and comparative examples were confirmed to possess fructosyl amino acid oxidase activity with F-Lys as a substrate. Therefore, their activities against F-Val-His, F-Val, and F-Gly were measured under the same conditions as for F-Lys. The specific activities of the wild-type and comparative examples against F-Lys, F-Val-His, F-Val, and F-Gly are shown in the table below. When the reactivity to F-Lys was set to 100, the reactivity of the wild-type fructosyl amino acid oxidase (serial number 1) against F-Val-His, F-Val, and F-Gly was less than 1. This confirms that the wild-type fructosyl amino acid oxidase exhibits F-Lys-specific reactivity.

[0147] [Table 13]

[0148]

[0149] 2. Activity and thermal stability of fructose-based amino acid oxidase in the embodiment where it is not fixed to a support. Analysis

[0150] The following describes the detailed production method of the fructose-based amino acid oxidase used in this embodiment. First, a gene containing a base sequence encoding the amino acid sequence described in this specification and designed by adding a histidine tag to the N-terminus of each inserted protein was introduced into an expression vector having a lactose operon. This expression vector was then used to transform Escherichia coli BL21(DE3) Derived strain (manufactured by Nippon Gene Co., Ltd.).

[0151] Transformed *E. coli* were cultured with shaking for 2 hours in LB liquid medium containing 50 μg / mL kanamycin (manufactured by Nakalitesk). These *E. coli* were then cultured at 16°C for 20 hours in LB liquid medium supplemented with isopropyl β-D-thiogalactoside (hereinafter referred to as "IPTG") to a final concentration of 0.1 mM. The collected *E. coli* were then suspended in buffer A (20 mM Tris-HCl (pH 7.5) containing 0.25 M sodium chloride and 20 mM imidazole), and the cells were disrupted using an ultrasonic disruptor. The cells were then centrifuged to obtain the cell lysate supernatant. After processing the cell lysate supernatant through a 0.45 μm filter, it was loaded into an EconoFit Nuvia IMAC Column, Ni-charged (BIO-RAD). The column was washed with 7 times its capacity 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).

[0152] The resulting eluent was dialyzed against a 10 mM sodium phosphate buffer (pH 8.4) containing 150 mM sodium chloride and 50 v / v glycerol to obtain the sample. The protein concentration in the sample was determined using absorbance at 280 nm.

[0153] The activity and thermostability of FAOD obtained by substituting amino acids in the amino acid sequence shown in Serial No. 1 (hereinafter referred to as "Examples") and FAOD containing the amino acid sequence shown in Serial No. 1 (hereinafter referred to as "wild-type") were evaluated. 50 μL of HEPES buffer (hereinafter referred to as "enzyme dilution") containing 0.01–0.1 mg / mL of each enzyme was dispensed into tubes and incubated on ice for 15 minutes or on a heating block at 45°C for 15 minutes. Then, 0.5 w / w% TOOS, POD / 4-AA solution (diluted with HEPES buffer (pH 8.0, 0.15 M NaCl) to the wells of a microplate containing 1 mM F-Lys at room temperature, along with the enzyme dilution (incubated on ice or heat-treated on a heating block), were added and mixed by pipetting. Using a microplate reader set to 37°C, absorbance was measured at 555 nm every 80 seconds after addition for 20 minutes. The change in absorbance per unit time was calculated as FAOD activity. Hereinafter, the activity measured using enzymes maintained on ice will be referred to as "FAOD activity before heat treatment", and the activity measured using enzymes heat-treated with a heating block will be referred to as "FAOD activity after heat treatment".

[0154] Using the average activity of the examples and wild-type FAOD, the activity improvement rate and thermal stability improvement rate before and after heat treatment were calculated by the following mathematical formula.

[0155]

[0156]

[0157]

[0158] The improvement rates of enzyme activity and thermal stability before and after heat treatment in the examples are shown below. Compared with wild-type enzymes, the enzymes of the examples showed at least one effect of improved activity before heat treatment, improved activity after heat treatment, and improved thermal stability.

[0159] [Table 14]

[0160]

[0161] In the table, "-" indicates that the enzyme activity after heat treatment was not confirmed, and therefore no value could be calculated.

[0162] Furthermore, based on the above results, examples with two or more amino acid substitutions were prepared, and their enzyme activity and thermostability were evaluated. The prepared examples were mutants with N5L and G225N amino acid substitutions relative to the amino acid sequence shown in Serial No. 1. The evaluation method was as described above. As shown below, compared with mutants having only N5L and G225N amino acid substitutions, a significant increase in activity and thermostability after heat treatment was confirmed.

[0163] [Table 15]

[0164]

[0165] In another embodiment, the activity and thermal stability of the enzyme were evaluated in its unsupported state. The evaluation method was as described above, with the heat treatment set to 45°C. The activity enhancement rate of the enzyme after heat treatment in the embodiment is shown below. Compared to the wild-type enzyme, the heat treatment confirmed an increase in activity in the enzyme of the embodiment.

[0166] [Table 16]

[0167]

[0168] Next, another modified FAOD was prepared to confirm whether its activity and thermostability were improved. This other FAOD used a FAOD containing the amino acid sequence shown in SEQ ID NO. 199. This FAOD is a fructosyl amino acid oxidase from Aspergillus fumigatus [Accession Number: AAB88209], also known as Amadoxin I. The amino acid sequence of this FAOD was confirmed to have 79.2% homology with the amino acid sequence shown in SEQ ID NO. 1.

[0169] The FAOD and its modified forms were prepared using the same method described above, and the activity enhancement rates before heat treatment, the activity enhancement rates after heat treatment, and the thermal stability enhancement rates were evaluated. As shown in Table 17, it was confirmed that the modified forms obtained by replacing methionine at position 58 with other amino acids exhibited at least one of the following effects compared to the wild-type enzyme: increased activity before heat treatment, increased activity after heat treatment, and improved thermal stability.

[0170] [Table 17]

[0171]

[0172] 3. The activity and thermal stability of fructose-based amino acid oxidase in the embodiment where it is fixed to a support. analyze

[0173] For the fructosyl amino acid oxidases of the examples and wild-type, the activity and thermostability in the immobilized state were evaluated. Bovine serum albumin (BSA) was used as the support. 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, with glutaraldehyde added to achieve a concentration of 0.1% (w / v). After incubation at 25°C for 20 min, 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 stopped by adding excess Tris containing primary amines. Enzyme activity was measured after maintaining the reaction at 15°C on ice or at 45°C on a heating block for 15 min. The rate of increase in activity before and after heat treatment, as well as the rate of increase in thermostability, were calculated using the average FAOD activity of each enzyme.

[0174] The activity enhancement rate and thermal stability enhancement rate of the enzymes in the examples before and after heat treatment are shown below. Compared with wild-type enzymes, the enzymes of the examples immobilized on the support showed at least one effect of increased activity before heat treatment, increased activity after heat treatment, and improved thermal stability.

[0175] [Table 18]

[0176]

[0177] Furthermore, the activity and thermal stability of the enzyme in the embodiment immobilized on the support were evaluated. The evaluation method was as described above, with the heat treatment set to 45°C. The rate of increase in activity and the rate of increase in thermal stability of the enzyme before and after heat treatment are shown below. Compared with the wild-type enzyme, the enzyme of the embodiment immobilized on the support showed at least one effect of increased activity before heat treatment, increased activity after heat treatment, and increased thermal stability.

[0178] [Table 19]

[0179]

[0180] In addition to the examples and wild type, comparative examples (FAOD-E, manufactured by Kikkoman Co., Ltd.) were used to evaluate the activity and thermal stability when immobilized on a support. Bovine serum albumin (BSA) was used as the support, and activity was determined using the method described above. The heat treatment temperature was set to 45°C. The residual activity rate and the improvement rate of thermal stability after heat treatment were calculated using the following mathematical formulas.

[0181]

[0182]

[0183] The following table shows the residual activity rates of the comparative examples and wild type, and the improvement rates of residual activity rates and thermal stability of the examples. A significant improvement in thermal stability was confirmed in each example compared to the wild type.

[0184] [Table 20]

[0185]

[0186] 4. Evaluation of sensors possessing wild-type and modified fructose-based amino acid oxidases

[0187] Glycated protein sensors with wild-type and modified FAOD were fabricated, and their thermal stability was evaluated. The wild-type FAOD used was the FAOD with the amino acid sequence shown in SEQ ID NO. 1, and the modified FAOD used was the FAOD with the amino acid substitution M58F in the wild-type FAOD, i.e., the FAOD with the amino acid sequence shown in SEQ ID NO. 38. A platinum electrode was silane-coupled to introduce amino groups onto the electrode surface. The FAOD was then added to a TES buffer containing bovine serum albumin (BSA), stirred until dissolved, and impurities were removed by centrifugation and filtration to prepare an enzyme solution. A predetermined amount of glutaraldehyde solution was added to this enzyme solution and stirred immediately. 1 μL of this solution was added dropwise to the amino-introduced platinum electrode, and the electrode was thoroughly dried to obtain a sensor containing FAOD and BSA as a support. Four sensors were fabricated for both wild-type and modified FAOD, resulting in a total of eight sensors.

[0188] For each sensor, 200 μL of HEPES buffer containing 50 μM F-Lys was added, and the change in current value was recorded as the sensor's output value. After cleaning with HEPES buffer, each sensor was dried with a hairdryer and allowed to stand at 75°C and 60% relative humidity for a certain period. Afterward, the sensors were removed, and their output values ​​were recorded using the same method. For the same sensor, this process of resting and recording output values ​​was repeated on days 1, 2, 3, 6, and 7, recording the output values ​​for each day. The evaluation results are presented below. Figure 2 The results showed that the sensor with modified FAOD had significantly higher output values ​​from day 1 to day 7 compared to the sensor with wild-type FAOD, and the rate of change was also significantly higher from day 1 to day 7 when day 0 was set to 100%.

[0189] That is, the sensor in this embodiment is a sensor having a support body and a FAOD modified body fixed to the support body. After the sensor is left to stand for 1 day at 75°C and 60% relative humidity, it maintains 73% of the output value. After the sensor is left to stand for 2 days under the same conditions, it maintains 61% of the output value. After the sensor is left to stand for 3 days under the same conditions, it maintains 49% of the output value. After the sensor is left to stand for 6 to 7 days under the same conditions, it maintains 27% of the output value.

[0190] Several embodiments and examples of the present invention have been described above, but these embodiments and examples are illustrative of the present invention. For example, the above embodiments are described in detail for ease of understanding of the present invention, and the size, structure, material, and circuitry may be added or changed as needed. It should be noted that embodiments formed by arbitrarily combining one or more features of the present invention listed above are also included within the scope of the present invention. The claims include various modifications to the embodiments without departing from the technical concept of the present invention. Therefore, the embodiments and examples disclosed in this specification are for illustrative purposes only and should not be considered as limiting the scope of the present invention.

Claims

1. A fructosyl amino acid oxidase having an amino acid sequence in which 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 Serial No. 1 is substituted when compared with the amino acid sequence shown in Serial No.

1.

2. The fructosyl amino acid oxidase according to claim 1, wherein, Compared with fructosyl amino acid oxidases that have not undergone amino acid substitution, fructosyl amino acid oxidases have higher activity and / or thermal stability.

3. The fructosyl amino acid oxidase according to claim 2, wherein, Compared with fructosyl amino acid oxidases that have not undergone amino acid substitution, fructosyl amino acid oxidases have more than 4% higher activity and / or thermal stability.

4. The fructose-based amino acid oxidase according to claim 2, wherein, The activity and / or thermal stability refer to the activity and / or thermal stability of the fructose-based amino acid oxidase when it 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 was higher than that of the fructosyl amino acid oxidase that had not undergone amino acid substitution after heat treatment.

6. The fructosyl amino acid oxidase according to claim 5, wherein, The heat treatment conditions are 40°C or 48°C for 15 minutes.

7. The fructosyl amino acid oxidase according to claim 1, wherein, Compared with the fructosyl amino acid oxidase before amino acid replacement, it exhibits higher thermal stability after heat treatment at 40°C or 48°C for 15 minutes.

8. The fructose-based amino acid oxidase according to any one of claims 1 to 7 has higher thermal stability when immobilized on a support than when not immobilized on a support.

9. The fructose-based amino acid oxidase according to claim 8, wherein, The fructosyl amino acid oxidase exhibits fructosyl lysine-specific reactivity.

10. The fructosyl amino acid oxidase according to claim 9, wherein, The reactivity refers to the fact that, when the reactivity to fructosyl lysine is set to 100, the reactivity to fructosyl valine, peptides containing fructosyl valine, and fructosyl glycine is below 20.

11. The fructosyl amino acid oxidase according to claim 2, having an amino acid sequence that shows more than 75% homology with the amino acid sequence shown in Serial No.

1.

12. The fructosyl amino acid oxidase according to claim 11, having an amino acid sequence that satisfies at least any one of (1) to (9) below, (1) The amino acid corresponding to the 5th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: 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 the 58th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: 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 the 59th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: 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 the 98th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: 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 the 225th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: 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 the 277th position of the amino acid sequence shown in sequence number 1 is any one of the following groups: 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 the 285th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: 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 the 355th position of the amino acid sequence shown in sequence number 1 is selected from any one of the following groups: 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 the 440th position of the amino acid sequence shown in sequence number 1 is any one of the following groups: 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 showing more than 79% homology with the amino acid sequence shown in Serial No.

1. When compared with the amino acid sequence shown in Serial No. 1, the amino acid corresponding to position 58 in the amino acid sequence shown in Serial 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, comprising: The amino acid sequence shown in any of the sequences 2 to 198 and 200 to 218; or An amino acid sequence formed by deleting, substituting, adding and / or inserting 1 to 5 amino acids in the amino acid sequence.

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

17. An expression vector containing the gene of claim 16.

18. A glycated protein sensor comprising the fructosyl amino acid oxidase of 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 fructose-based amino acid oxidase is immobilized on the support by cross-linking with an amine-reactive cross-linking agent.

20. The sensor according to claim 19, wherein, The output value of the sensor after standing at 65°C or 80°C for 20 minutes is more than 60% of the output value of the sensor before standing; or, the output value of the sensor after standing at 75°C and 60% relative humidity for 1 day is more than 40% of the output value of the sensor before standing.

21. A method for determining glycated proteins, wherein the glycated proteins are detected by the reaction of the fructosyl amino acid oxidase as described in claim 2.

22. A method for manufacturing a fructosyl amino acid oxidase, comprising the following amino acid substitution step: for the amino acid sequence of the fructosyl amino acid oxidase, substituting any amino acid corresponding to a position in the group consisting of the 5th, 58th, 225th, 277th and 440th positions in the amino acid sequence shown in Serial No. 1 when the amino acid sequence is compared with the amino acid sequence shown in Serial No.

1.

23. The method for manufacturing fructosyl amino acid oxidase according to claim 22, wherein, The amino acid sequence of the fructose-based amino acid oxidase is an amino acid sequence that has more than 79% homology with the amino acid sequence shown in sequence number 1.

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