MODIFIED AMADORIASE AND METHOD FOR MANUFACTURING THE SAME AS WELL AS AGENT FOR IMPROVING SURFACTANT RESISTANCE OF AMADORIASE AND COMPOSITION FOR MEASURING HbA1c USING THE SAME

Abstract

To provide a composition that allows the measurement of glycated hemoglobin even in the presence of stronger surfactants than conventional ones, and to provide a buffer and / or stabilizer that maintains the residual activity of amadoriase or reduces the decrease in the residual activity.SOLUTION: The present invention provides a composition for measuring glycated hemoglobin, comprising an amadoriase having an amino acid residue substitution at a position corresponding to an amino acid selected from the group consisting of 262, 257, 249, 253, 337, 340, 232, 129, 132, 133, 44, 256, 231, and 81 of an amadoriase derived from the genus Coniochaeta shown in a specific sequence, and an amadoriase that retains activity even in the presence of a surfactant. The present invention provides a composition and kit for measuring glycated hemoglobin that contain a specific stabilizer and / or buffer, and can provide an enzyme and a composition for measuring glycated hemoglobin that have excellent storage stability even in the presence of a surfactant.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention can be advantageously used as an enzyme for diagnosing diabetes and in a kit for measuring diabetes markers. Amadoriase with excellent surfactant resistance that can be used, its gene and recombinant DNA, etc. The present invention also relates to a method for producing an amadoriase having excellent resistance to surfactants. The present invention relates to a lyase stabilizer and / or buffer, and to compositions containing the same. [Background technology]

[0002] Glycated proteins are composed of aldoses (monosaccharides potentially containing an aldehyde group) such as glucose. The aldehyde group of α-aminobutyric acid (α-aminobutyric acid or its derivatives) and the amino group of a protein form a covalent bond nonenzymatically. The amino group of the protein is Examples include the α-amino group at the amino terminus and the ε-amino group of the lysine residue side chain in a protein. Glycated proteins that occur in the body include hemoglobin in the blood, which is glycosylated, called glycated hemoglobin. Examples of albumin that has been glycosylated include glycated albumin and glycated albumin.

[0003] Among these glycated proteins that occur in the body, diabetes mellitus is one of the most common glycated proteins in the field of clinical diagnosis of diabetes. Glycohemoglobin (GHC) is an important glycemic control marker for the diagnosis and symptom management of patients with hemoglobinemia. The HbA1c concentration in blood is the average for a certain period of time in the past. It reflects blood glucose levels, and its measurement is an important indicator in diagnosing and managing diabetes symptoms. It is.

[0004] As a method for quickly and easily measuring HbA1c, an enzymatic method using amadoriase has been proposed. That is, HbA1c is decomposed with protease or the like and released from the amino terminal of the β chain. α-Fructosyl valyl histidine (hereinafter referred to as "αFVH") or α-fructosyl valyl histidine (hereinafter referred to as "αFVH") Methods for quantifying αFV have been proposed (e.g., (See Patent Documents 1 to 7.) In fact, in the method of isolating αFV from HbA1c, However, there is a problem that accurate measurement values ​​cannot be obtained due to the large influence of factors such as In order to obtain this information, measuring αFVH has become the mainstream method, especially at present.

[0005] Amadoriase reacts with iminodiacetic acid or its derivatives ("amadoriase") in the presence of oxygen. The compound is oxidized to glyoxylic acid or α-ketoaldehyde, amino acid or catalyzes a reaction that produces a peptide and hydrogen peroxide.

[0006] Amadoriase has been found in bacteria, yeast, and fungi, but is particularly useful for measuring HbA1c. and amadoriase having enzymatic activity against αFVH and / or αFV, Examples of such fungi include the genus Coniochaeta, Eupenicillium, penicillium genus, Pyrenochaeta genus, Arthrix Genus Arthrinium, Curvularia, Neoco Neocosmospora, Cryptococcus cus), Phaeosphaeria, Aspergillus (A spergillus, Emericella, Urocladium (Ulocladium), Penicillium, Fusarium Fusarium genus, Achaetomiella genus, Achaetomiella Achaetomium spp., Thielavia spp. Genus Chaetomium, Gelasinospora, Genus Microascus, Leptosphaeria ria), Ophiobolus, Pleospora ora genus, Coniochaetidium genus, Pichia hia genus, Corynebacterium genus, Agrobacterium Genus Agrobacterium, Arthrobacter Amadoriases derived from the genus Amadoria have been reported (for example, Patent Documents 1, 6 to 15, and Non-Patent Documents 1 and 2). (See References 1 to 11.) In the above reported cases, amadoriase is not considered to be a fructosylamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase It may also be described as fructosylamine oxidase, fructosylamine oxidase, etc.

[0007] When measuring HbA1c, the reagent composition for the measurement contains an excess amount of amadoriase. For example, when measuring HbA1c at a final concentration of 0.36 μM, Amadoriase reacts with 1.4 kU / L, i.e., 1.4 mM of substrate per minute. The concentration used is such that it is possible to obtain Hb using amadoriase (see Patent Document 16). The measurement of A1c is mainly done using an automatic analyzer. The reaction time with the substrate is usually about 5 to 25 minutes. The reason for including the amine in the above is that it is necessary to react sufficiently with the substrate within the short measurement time described above. In addition, there are substances that can have a significant negative effect on the reactivity and stability of amadoriase. When amadoriase coexists in the measurement composition, it is necessary to use an excess of amadoriase to prevent this effect. This is because they have no choice but to be mixed together.

[0008] A pretreatment method for measuring HbA1c from whole blood or red blood cells using amadoriase Examples of hemolysis using surfactants include those in Patent Documents 2 and 16. 18.) When HbA1c is decomposed by protease, surfactants act as reaction promoters. In some cases, agents are used (see, for example, Patent Document 19). When measuring HbA1c using a surfactant, surfactants are indispensable. The HbA1c solution treated with the drug and protease was mixed with the amadoriase solution, and then H When starting the bA1c quantitative reaction, and when mixing and storing the surfactant and amadoriase, When the surfactant is used, it is highly likely that the amadoriase is denatured. The c measurement kit contains more amadoriase than necessary and a stabilizer. Although accurate measurements can be achieved because of the large amount of reagents used, the cost is high. If it were possible to use a surfactant that is more effective than the current one, it would be possible to reduce Hb The efficiency of protease degradation of A1c may be improved, which may improve the sensitivity of HbA1c measurement. In addition, surfactants have a high solubility in water and can dissolve insoluble peptide fragments derived from hemoglobin and HbA1c. It also has a solubilizing effect, which prevents turbidity and contributes to improving measurement accuracy. The properties required for formulating amadoriase into a kit reagent as an enzyme for clinical diagnosis of diabetes mellitus One of its qualities is the good stability of the liquid form containing surfactants.

[0009] Although the actual measurement conditions vary, there are many published literature on the measurement of amadoriase in liquid form. Stability disclosure: Coniochaeta sp. NISL 9330 The solution containing the amadoriase from the strain was supplemented with 5 mM ethylenediaminetetraacetic acid and 3% glucose. When lysine was added, 79% of the residual activity was maintained after 7 days at 30°C. (For example, see Patent Document 20). Also, Fusarium oxysp orum IFO-9972 strain-derived fructosyl amino acid oxidase solution, When 3% L-alanine, 3% glycine, or 3% sarcosine was added, the It has been shown that the drug maintains 100% residual activity after 2 days (see, for example, See reference 21. ).

[0010] However, the above-mentioned solution containing amadoriase protein does not contain any surfactant. No surfactants are added, and there is no description of reducing the effects of surfactants. There have been no reports of amadoriase with high activity in the presence of detergents. No stabilizers or buffers have been reported that maintain residual activity or reduce the loss of residual activity. . [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2004 / 104203 [Patent Document 2] International Publication No. 2005 / 49857 [Patent Document 3] JP 2001-95598 A [Patent Document 4] Special Publication No. 05-33997 [Patent Document 5] Japanese Patent Application Publication No. 11-127895 [Patent Document 6] International Publication No. 97 / 13872 [Patent Document 7] JP 2011-229526 A [Patent Document 8] JP 2003-235585 A [Patent Document 9] JP 2004-275013 A [Patent Document 10] JP 2004-275063 A [Patent Document 11] JP 2010-35469 A [Patent Document 12] JP 2010-57474 A [Patent Document 13] International Publication No. 2010 / 41715 [Patent Document 14] International Publication No. 2010 / 41419 [Patent Document 15] International Publication No. 2011 / 15325 [Patent Document 16] International Publication No. 2012 / 020744 [Patent Document 17] WO 2005 / 87946 [Patent Document 18] International Publication No. 2002 / 06519 [Patent Document 19] International Publication No. 2006 / 120976 [Patent Document 20] JP 2006-325547 A [Patent Document 21] JP 2009-000128 A [Non-patent literature]

[0012] [Non-Patent Document 1] Biochem. Biophys. Res. Commun. 311, 104-11, 2003 [Non-Patent Document 2] Biotechnol. Bioeng. 106, 358-66, 2010 [Non-Patent Document 3] J. Biosci. Bioeng. 102, 241-3, 2006 [Non-Patent Document 4] Eur. J. Biochem. 242, 499-505, 1996 [Non-Patent Document 5] Arch.Microbiol.178,344-50,2002 [Non-Patent Document 6] Mar. Biotechnol. 6, 625-32, 2004 [Non-Patent Document 7] Biosci. Biotechnol. Biochem.59, 487-91,1995 [Non-Patent Document 8] Appl. Microbiol. Biotechnol. 74, 813-819, 2007 [Non-Patent Document 9] Biosci. Biotechnol. Biochem. 66, 1256-61, 2002 [Non-Patent Document 10] Biosci. Biotechnol. Biochem. 66, 2323-29, 2002 [Non-Patent Document 11] Biotechnol. Letters 27, 27-32,2005 Summary of the Invention [Problem to be solved by the invention]

[0013] As described above, conventionally, amadoriase is required to react sufficiently with the substrate during the measurement time. The present inventors have found that surfactants have a significant negative effect on the stability of amadoriase. Therefore, it has been newly discovered that amadoriase is a component that can affect the If an enzyme with better surfactant resistance than the above is produced, the enzyme and the kit will be more efficient. By reducing the amount of amadoriase and stabilizer in the kit, Improved sensitivity of HbA1c measurement due to lower costs and the ability to formulate strong surfactants It is expected that the present invention will make a significant contribution to the field of the present invention. To provide an amadoriase having superior surfactant resistance compared with conventional amadoriases The quantification of HbA1c or glycated peptides derived from HbA1c was possible even in the presence of surfactants. The present invention aims to provide a possible reagent composition.

[0014] Another problem to be solved by the present invention is the persistence of amadoriase in the presence of a surfactant. Stabilizing agents and / or buffers that maintain activity or reduce the decrease in residual activity, and The present invention relates to a composition comprising: [Means for solving the problem]

[0015] Currently, there is almost no information available on how to give enzymes surfactant resistance. As a result of extensive research, the present inventors have discovered amadoriase derived from the genus Coniochaeta. By introducing specific amino acid substitutions, the The above problems can be solved by including amadoriase that still has activity in the reagent composition. It was also found that the use of a specific stabilizer and / or buffer reduces the presence of a surfactant. The residual activity of amadoriase can be maintained or the decrease in the residual activity can be significantly reduced under the above conditions. The present invention has been completed based on this finding.

[0016] That is, the present invention is as follows.

[0017] [1] The residual activity (%) 5 minutes after addition of the surfactant was 7, which is an improved amadoriase compared to the amadoriase having the amino acid sequence shown in There was, (i) a deletion of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1, 3, or 37; , an amino acid sequence having insertions, additions, and / or substitutions; and / or (ii) at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 1, 3, or 37; having an amino acid sequence The amadoriase.

[0018] [2] The amadoriase described in [1], wherein the surfactant is an ionic surfactant.

[0019] [3] The amino acid sequence of SEQ ID NO: 1 or 3, which is selected from the following (i) to (xiv): (i) asparagine at position 262, (ii) valine at position 257; (iii) glutamic acid at position 249 (iv) glutamic acid at position 253, (v) glutamine at position 337; (vi) glutamic acid at position 340, (vii) aspartic acid at position 232, (viii) aspartic acid at position 129, (ix) aspartic acid at position 132, (x) glutamic acid at position 133, (xi) glutamic acid at position 44, (xii) glycine at position 256, (xiii) glutamic acid at position 231, and (xiv) glutamic acid at position 81, one or more amino acid residues at positions corresponding to amino acids selected from the group consisting of The amadoriase according to [1] or [2], having the following substitution:

[0020] [4] The amino acid sequence shown in SEQ ID NO: 1 or 3 is Substitutions (i) to (xiv) below: (i) substitution of asparagine at position 262 with histidine; (ii) substitution of valine at position 257 with cysteine, serine, or threonine; (iii) substitution of glutamic acid at position 249 with lysine or arginine; (iv) substitution of glutamic acid at position 253 with lysine or arginine; (v) substitution of glutamine at position 337 with lysine or arginine; (vi) substitution of glutamic acid at position 340 with proline; (vii) substitution of aspartic acid at position 232 with lysine or arginine; (viii) substitution of aspartic acid at position 129 with lysine or arginine; (ix) substitution of aspartic acid at position 132 with lysine or arginine; (x) glutamic acid at position 133 is replaced by alanine, methionine, lysine, or arginine are; (xi) glutamic acid at position 44 is replaced by proline; (xii) substitution of glycine at position 256 with lysine or arginine; (xiii) glutamic acid at position 231 is replaced with lysine or arginine; and (xiv) glutamic acid at position 81 is replaced with lysine or arginine; The amadoriase according to any one of [1] to [3], having one or more of the following:

[0021] [5] In the amino acid sequence shown in SEQ ID NO: 1 or 3, the following (i) to (ix): (i) Substitution of the amino acid at position 44 corresponding to glutamic acid with proline and substitution of the amino acid at the position corresponding to glutamic acid at position 1 with proline; (ii) substitution of the amino acid at position 44 corresponding to glutamic acid with proline, Substitution of the amino acid at the position corresponding to asparagine with histidine and substitution of glutamine at position 340 Substitution of the amino acid at the position corresponding to carboxylic acid with proline; (iii) substitution of the amino acid at the position corresponding to glutamic acid at position 44 with proline; substitution of the amino acid at position 262 corresponding to valine with cysteine; Substitution of the amino acid at the corresponding position with histidine and the amino acid at the corresponding position with glutamic acid at position 340 Substitution of amino acid 1 at position 4 with proline; (iv) substitution of the amino acid at position 44 corresponding to glutamic acid with proline, Substitution of the amino acid at the position corresponding to valine with cysteine, corresponding to asparagine at position 262 and substitution of the amino acid at position 340 corresponding to glutamic acid with histidine. The amino acid at position 232 was replaced by proline and the amino acid at position 233 was replaced by aspartic acid at position 232. Substitution for lysine; (v) substitution of the amino acid at position 44 corresponding to glutamic acid with proline; Substitution of the amino acid at the position corresponding to valine with cysteine, corresponding to asparagine at position 262 and substitution of the amino acid at position 340 corresponding to glutamic acid with histidine. The amino acid at position 249 was replaced by proline and the amino acid at position 250 was replaced by glutamic acid at position 249. Substitution for gin; (vi) substitution of the amino acid at position 44 corresponding to glutamic acid with proline; Substitution of the amino acid at the position corresponding to glutamic acid with lysine, corresponding to valine at position 257 substitution of the amino acid at position 262 with cysteine; substitution of histidine for 340 and the amino acid protease corresponding to glutamic acid at position 340. Substitution for phosphorus; (vii) substitution of the amino acid at the position corresponding to glutamic acid at position 44 with proline; substitution of the amino acid at position 257 corresponding to glutamic acid with lysine; substitution of the amino acid at position 262 with cysteine; substitution of 340 glutamic acid for histidine; and substitution of the amino acid at position 129 corresponding to aspartic acid at position 129 with lysine. ; (viii) substitution of the amino acid at the position corresponding to glutamic acid at position 44 with proline; substitution of the amino acid at the position corresponding to glutamic acid in position 253 with alanine; substitution of the amino acid at position 257 corresponding to valine with lysine; and a substitution of histidine at the amino acid position corresponding to asparagine at position 262. and substitution of the amino acid at position 340 corresponding to glutamic acid with proline. ;and (ix) substitution of the amino acid at position 44 corresponding to glutamic acid with proline, Substitution of the amino acid at the position corresponding to glutamic acid with alanine, glutamic acid at position 253 Substitution of the amino acid at the corresponding position with lysine, the amino acid at the position corresponding to valine at position 257 to cysteine, and histidine at the amino acid position corresponding to asparagine at position 262. substitution of the amino acid at the position corresponding to glutamine at position 337 to lysine; Substitution of the amino acid at the position corresponding to glutamic acid at position 0 with proline [4] Any of [1] to [4], having a substitution of an amino acid residue selected from the group consisting of: Amadoriase.

[0022] [6] The amino acid sequence of SEQ ID NO:37 is Substitutions (i) to (ix) below: (i) glutamic acid at position 247 is replaced by lysine or arginine; (ii) substitution of glutamic acid at position 251 with lysine or arginine; (iii) substitution of threonine at position 335 with lysine or arginine; (iv) substitution of aspartic acid at position 230 with lysine or arginine; (v) substitution of aspartic acid at position 129 with lysine or arginine; (vi) substitution of aspartic acid at position 132 with lysine or arginine; (vii) glutamic acid at position 133 is replaced with alanine, methionine, lysine, or arginine Being; (viii) asparagine at position 254 is substituted with lysine or arginine; and (ix) substitution of glutamic acid at position 229 with lysine or arginine; The amadoriase according to any one of [1] to [3], having one or more of the following:

[0023] [7] In the amino acid sequence shown in SEQ ID NO: 37, the following (i) to (iv) are selected from the group consisting of: (i) Substitution of the amino acid at the position corresponding to glutamic acid at position 251 with lysine and substitution of lysine for the amino acid at the position corresponding to threonine at position; (ii) Substitution of the amino acid at the position corresponding to aspartic acid at position 132 with lysine and Substitution of the amino acid at the position corresponding to threonine at position 5 with lysine; (iii) Substitution of the amino acid at the position corresponding to glutamic acid at position 133 with alanine and A substitution of the amino acid at the position corresponding to threonine at position 35 with lysine; and (iv) Substitution of the amino acid at the position corresponding to glutamic acid at position 229 with lysine and substitution of the amino acid at the position corresponding to the threonine at position 2 with lysine; Any of [1] to [3] and [6], wherein the amino acid residue is substituted with a substituent selected from the group consisting of: The amadoriase described above.

[0024] [8] An amadoriase gene encoding the amino acid sequence according to any one of [1] to [7].

[0025] [9] A recombinant vector containing the amadoriase gene described in [8].

[0026]

[10] A host cell containing the recombinant vector described in [9].

[0027]

[11] The process involves: (i) culturing the host cell according to

[10] ; (ii) expressing the amadoriase gene contained in the host cell; and (iii) producing amadoriase, comprising the step of isolating the amadoriase from the culture. method.

[0028]

[12] A method for measuring glycated hemoglobin, comprising the amadoriase according to any one of [1] to [7]. A composition for use in

[0029]

[13] A composition for measuring glycated hemoglobin, comprising one or more surfactants and amadoriase. .

[0030]

[14] Amadoriase (i) The residual activity (%) 5 minutes after adding the surfactant was compared to that without the surfactant. , 15% or more remaining, and / or (ii) In the presence of a detergent at a final concentration of 0.04%, the chromogenic substrate N-(carboxymethyl) (dimethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine sodium After adding glycated amine (DA-64) and reacting for 5 minutes, the absorbance at 751 nm and the Five minutes after addition of the control solution, which used ion-exchanged water instead of the acid solution or glycated peptide solution The difference between the absorbance at 751 nm of

[13] The composition described in.

[0031]

[15] Amadoriase has at least one amino acid sequence represented by SEQ ID NO: 1, 3, 37 or 40. The composition described in

[13] or

[0014] , wherein the amadoriase has an amino acid sequence that is 70% identical to both of the amadoriases.

[0032]

[16] The composition described in any one of

[13] to

[0015] , wherein the surfactant has a critical micelle concentration of 70 mM or less.

[0033]

[17] The surfactant is a quaternary ammonium salt represented by the following general formula (I): [ka] [In the formula, R 1 ~R 4 may be the same or different, and may be substituted or unsubstituted C1 to C2 0 represents alkyl, alkenyl, aryl or benzyl; Z - represents a monovalent anion .] A pyridinium salt represented by the following general formula (II): [ka] [In the formula, R 5 is a substituted or unsubstituted C1-C 20 Each R represents an alkyl group. a is the same or may be different, and each represents a hydrogen atom or a substituted or unsubstituted C1-C 20 Alkyl, ar represents an aryl, aryl or benzyl; n represents an integer of 1 to 5; Z - is a monovalent anion ] A phosphonium salt represented by the following general formula (III): [ka] [In the formula, R 6 ~R 9 may be the same or different, and may be substituted or unsubstituted C1 to C2 0 represents alkyl, alkenyl, aryl or benzyl; Z - represents a monovalent anion .] and one or more ionic surfactants selected from the group consisting of sodium dodecyl sulfate. The composition according to any one of

[13] to

[16] .

[0034]

[18] The surfactant is octyltrimethylammonium chloride, octyltrimethyl Ammonium bromide, dioctyl dimethyl ammonium chloride, dioctyl dimethyl Ammonium bromide, decyltrimethylammonium chloride, decyltrimethylan Dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium ammonium bromide, tetradecyltrimethylammonium chloride, tetradecyltrimethylammonium Hexadecyl ammonium bromide, Hexadecyltrimethylammonium chloride, Hexadecyl Octadecyltrimethylammonium bromide, Octadecyltrimethylammonium chloride, Octadecyltrimethylammonium bromide, eicosyltrimethylammonium chloride ammonium bromide, eicosyltrimethylammonium bromide, benzyldodecyldimethylammonium Benzyldodecyldimethylammonium bromide, benzyltetradecyl Dimethylammonium chloride, benzyltetradecyldimethylammonium bromide, Benzylcetyldimethylammonium chloride, and benzylcetyldimethylammonium ium bromide, 1-Decylpyridinium chloride, 1-Decylpyridinium bromide, 1-Dodecylpyridinium pyridinium chloride, 1-dodecylpyridinium bromide, 1-tetradecylpyridinium 1-Tetradecylpyridinium bromide, 1-Hexadecylpyridinium chloride Bromide, 1-Hexadecylpyridinium bromide, N-Cetyl-2-methylpyridinium Chloride, N-cetyl-2-methylpyridinium bromide, N-cetyl-3-methylpyridinium pyridinium chloride, N-cetyl-3-methylpyridinium bromide, N-cetyl-4-methylpyridinium Cetylpyridinium chloride, N-cetyl-4-methylpyridinium bromide, 1-octylpyridinium chloride Decylpyridinium chloride, 1-octadecylpyridinium bromide, 1-eicosyl Pyridinium chloride and 1-eicosylpyridinium bromide, Tetraethylphosphonium chloride, Tetraethylphosphonium bromide, Tributyl Methylphosphonium chloride, Tributylmethylphosphonium bromide, Tributylmethyl butylphosphonium iodide, tetrabutylphosphonium chloride, tetrabutylphosphonium Bromide, Tetra-n-octylphosphonium chloride, Tetra-n-octylphosphonium nium bromide, tributyldodecylphosphonium chloride, tributyldodecylphosphonium nium bromide, tributylhexadecylphosphonium chloride, tributylhexadecyl methyltriphenylphosphonium bromide, methyltriphenylphosphonium chloride, methyltriphenyl methyltriphenylphosphonium bromide, methyltriphenylphosphonium iodide, tetraphenylphosphine phosphonium chloride, and tetraphenylphosphonium bromide, The composition according to

[17] , wherein the surfactant is one or more ionic surfactants selected from the group consisting of:

[0035]

[19] The surfactant contained in the solution must be less than 0.01% (w / v) as the final concentration at the time of measurement. The composition according to any one of

[13] to

[18] above.

[0036]

[20] Borate buffer, Tris-HCl buffer, phosphate buffer, citrate buffer, fumarate buffer Buffers, glutaric acid buffer, citraconic acid buffer, mesaconic acid buffer, malonic acid buffer, alcohol Tartaric acid buffer, succinic acid buffer, adipic acid buffer, ACES (N-(2-acetamido)-2-acetamide) N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffer, BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffer N,N-bis(2-hydroxyethyl)glycine) buffer, Bis-Tris (Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane) Buffer, EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid) buffer , HEPPSO (N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid ) buffer, MES (2-(N-morpholino)ethanesulfonic acid) buffer, MOPS (3-(N-morpholino)ethanesulfonic acid) buffer 2-Hydroxy-3-morpholinopropanesulfonic acid (MOPSO) buffer Sulfonic acid) buffer, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) buffer , POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid)) buffer, TA PS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer, TA PSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfone Acid) buffer, TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) Buffers, Tricine (N-tris(hydroxymethyl)methylglycine) buffers and combinations thereof The glycosylated hemoglobin concentration according to

[13] above further contains one or more buffers selected from the group consisting of Robin measuring composition.

[0037]

[21] Phosphate buffer with a final concentration of 100 mM or more in the measurement solution; Citrate buffer with a concentration of 10 mM or more, ME with a final concentration of 150 mM or more in the measurement solution S (2-(N-morpholino)ethanesulfonic acid) buffer, final concentration in the measurement solution is 100 mM or more MOPS (3-(N-morpholino)propanesulfonic acid) buffer, the final concentration of the measurement solution MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid) buffer with a concentration of 100mM or higher and ACES (N-(2-acetamido)-2-acetamide) at a final concentration of 200 mM or more in the measurement solution.

[20] The composition described in

[0038]

[22] Phosphoric acid, tricarboxylic acid, dicarboxylic acid, monocarboxylic acid, compound of formula (IV), [ka] [wherein n may be 0, 1, 2 or 3; R 10 are each independently H, OH, -CH2OH or -COOH] and one or more stabilizers selected from the group consisting of ammonium sulfate and combinations thereof. The composition for measuring glycated hemoglobin according to

[13] ,

[0039]

[23] The tricarboxylic acid is citric acid or the dicarboxylic acid is fumaric acid, glutathione acid, citraconic acid, mesaconic acid, malonic acid, tartaric acid, succinic acid, adipic acid, maleic acid or a combination thereof, selected from the group consisting of monocarboxylic acid, malic acid, and combinations thereof; The acid is acetic acid or the compound of formula (IV) is MES, MOPS, MOPSO and the like. The composition according to

[22] , wherein the composition is selected from the group consisting of:

[0040]

[24] The stabilizer is a phosphate having a final concentration of 2 mM or more in the measurement solution, Citric acid with a final concentration of 0.2 mM or more in the measurement solution, phosphorus with a final concentration of 2 mM or more in the measurement solution maleic acid, whose final concentration in the measurement solution is 2 mM or more; citraconic acid with a final concentration of 2 mM or more in the measurement solution, malonic acid with a final concentration of 2 mM or more in the measurement solution, Glutaric acid with a final concentration of 2 mM or more in the solution, and with a final concentration of 2 mM or more in the measurement solution Tartaric acid, acetic acid with a final concentration of 10 mM or more in the measurement solution, MES (2-(N-morpholino)ethanesulfonic acid) with a final concentration of 10 or more in the measurement solution The final concentration of MOPS (3-(N-morpholino)propanesulfonic acid) in the measurement solution is ≥ 1 mM. MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid) with a concentration of 10 mM or more Ammonium sulfate with a final concentration of 2 mM or more in the fixed solution, and combinations thereof The composition according to

[22] or

[23] , wherein the stabilizer is one or more selected from the group consisting of:

[0041]

[25] A buffer according to

[20] or

[21] and a stabilizer according to

[22] ,

[23] or

[24] . A composition for measuring glycated hemoglobin, comprising a stabilizing agent.

[0042]

[26] The amadoriase has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 37, or SEQ ID NO: 40. or the amadoriase according to any one of [1] to [7]. The composition according to any one of

[20] to

[25] .

[0043] This specification is a joint publication of Japanese Patent Application Nos. 2013-167005 and 2013-221515, which are the priority documents of this application. It encompasses what is described in the specification and / or drawings. Effect of the Invention

[0044] According to the present invention, the enzyme is useful as a diagnostic enzyme for diabetes and in a kit for measuring diabetes markers. US20130232633A1 - Detergent-resistant amadoriase that can be utilized for practical purposes and gene encoding same - Google Patents By using this amadoriase, it is possible to obtain a soluble form of cellulose in the presence of a high concentration of surfactant. The glycated hemoglobin can be measured even under low temperature. By using a buffer, the residual activity of amadoriase can be maintained in the presence of a detergent or This reduces the decrease in residual activity, making it possible to measure glycated hemoglobin even in the presence of high concentrations of surfactants. It can be done. [Brief description of the drawings]

[0045] [Figure 1-1] FIG. 1-1 is an alignment of the amino acid sequences of various known amadoriases. [Figure 1-2] FIG. 1-2 is an alignment of the amino acid sequences of various known amadoriases. [Figure 1-3] FIG. 1-3 shows an alignment of the amino acid sequences of various known amadoriases. [Diagram 2] FIG. 2 shows the results of measuring αFVH using CFP-T7 in the presence of 0.01% CTAC. [Diagram 3] FIG. 3 shows the results of measuring αFVH using CFP-T7 in the presence of 0.02% CTAC. [Figure 4] FIG. 4 shows the results of measuring αFVH using CFP-D7 in the presence of 0.02% CTAC. [Diagram 5] FIG. 5 shows the results of measuring αFVH using CFP-D7 in the presence of 0.2% CTAC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The present invention will be described in detail below.

[0047] (Amadoriase) Amadoriase is an enzyme that activates ketoamine oxidase, fructosyl amino acid oxidase, and fructosyl amino acid oxidase. It is also called fructosyl peptide oxidase or fructosyl amine oxidase. In the presence of Reactions that produce alkyl acids or α-ketoaldehydes, amino acids or peptides, and hydrogen peroxide Amadoriase is an enzyme that catalyzes the reaction. Amadoriase is widely distributed in nature and is found in microorganisms. These can be obtained by searching for enzymes of animal or plant origin. The enzyme can be obtained, for example, from filamentous fungi, yeasts, or bacteria.

[0048] One embodiment of the amadoriase of the present invention is a CoAmplification enzyme having the amino acid sequence shown in SEQ ID NO:1. Amadoriase from the genus niochaeta or the amino acid sequence shown in SEQ ID NO: 37 Based on the amadoriase from Curvularia clavata, In addition, it is a mutant of amadoriase that has improved resistance to detergents. Examples of such mutants include As such, a high sequence identity (e.g., 70% or more) with SEQ ID NO: 1 or SEQ ID NO: 37 is preferred. At most, 75% or more, preferably 80% or more, more preferably 85% or more, and even more preferably Preferably, the content is 90% or more, more preferably 95% or more, even more preferably 97% or more, and most preferably or 99% or more) of the amino acid sequence of SEQ ID NO: 1 or In the amino acid sequence of SEQ ID NO: 37, one to several amino acids are modified or mutated, or or an amadoriase having a deleted, substituted, added and / or inserted amino acid sequence. Examples include:

[0049] The amadoriase of the present invention can be, for example, from the genus Eupenicillium, Pyrenophilus spp. ochaeta sp., Arthrinium sp., Curvularia sp., Neocosm ospora, Cryptococcus, Phaeosphaeria, Asp ergillus, Emericella, Ulocladium, Penici Genus llium, Genus Fusarium, Genus Achaetomiella, Achaetom ium, Thielavia, Chaetomium, Gelasinospor Genus a, Microascus, Leptosphaeria, Ophiobolus Genus, Pleospora, Coniochaetidium, Pichia, Co rynebacterium genus, Agrobacterium genus, Arthrobact The enzyme may be based on amadoriase derived from a species of organism such as the genus E. coli. Among these, those having detergent resistance and / or having the amino acid sequence of SEQ ID NO: 1 or Preferably, the sequence has a high sequence identity with SEQ ID NO:37.

[0050] The mutant (modified) amadoriase with improved detergent resistance is Substituting or adding at least one amino acid residue in the amino acid sequence; and can be obtained by deletion.

[0051] As an amino acid substitution that improves detergent resistance, the amino acid shown in SEQ ID NO: 1 or 3 Substitutions of amino acids at positions corresponding to the following positions in the amino acid sequence are included: .

[0052] (1) Substitution of asparagine at position 262, for example, with histidine. (2) Substitution of valine at position 257, for example, with cysteine, serine, or threonine. (3) Substitution of glutamic acid at position 249, for example, with lysine or arginine. (4) Substitution of glutamic acid at position 253, for example, with lysine or arginine. (5) Substitution of glutamine at position 337, for example, with lysine or arginine. (6) Substitution of glutamic acid at position 340, for example, with proline. (7) Substitution of aspartic acid at position 232, for example, with lysine or arginine. (8) Substitution of aspartic acid at position 129, for example, with lysine or arginine. (9) Substitution of aspartic acid at position 132, for example, with lysine or arginine. (10) Substitution of glutamic acid at position 133, e.g., alanine, methionine, lysine, alginate, etc. Substitution for ginine. (11) Substitution of glutamic acid at position 44, for example, with proline. (12) Substitution of glycine at position 256, for example, with lysine or arginine. (13) Substitution of glutamic acid at position 231, for example, with lysine or arginine. (14) Substitution of glutamic acid at position 81, for example, with lysine or arginine.

[0053] The amadoriase mutants with improved detergent resistance have at least the above amino acid substitutions. It is sufficient to have one amino acid substitution, but it may also have multiple amino acid substitutions. having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 substitutions is.

[0054] Among these, preferred are mutants having amino acid substitutions corresponding to the following amino acid positions: I wish. (11)-(6) Substitution of glutamic acid at position 44 and substitution of glutamic acid at position 340, e.g., Substitution of the amino acid at position 44 corresponding to glutamic acid with proline and substitution of the amino acid at position 340 corresponding to glutamic acid with proline A mutant having a substitution of the amino acid at the position corresponding to glutamic acid with proline. (11)-(1)-(6) Substitution of glutamic acid at position 44, substitution of asparagine at position 262, and substitution of 34 Substitution of glutamic acid at position 0, e.g., of the amino acid at the position corresponding to glutamic acid at position 44 Substitution of proline at the amino acid position corresponding to asparagine 262 to histidine A mutation that substitutes the amino acid at the position corresponding to glutamic acid at position 340 for proline. body. (11)-(2)-(1)-(6) Substitution of glutamic acid at position 44, valine at position 257, Substitution of asparagine and substitution of glutamic acid at position 340, e.g., glutamine at position 44 substitution of the amino acid at position 257 corresponding to valine with proline; substitution of the amino acid at position 262 corresponding to asparagine at position 262 with cysteine; substitution of cysteine ​​to cysteine ​​and of the amino acid at the position corresponding to glutamic acid at position 340 to proline A variant having a substitution. (11)-(7)-(2)-(1)-(6) Substitution of glutamic acid at position 44, substitution of valine at position 257, substitution of asparagine at position 340, substitution of glutamic acid at position 232, and Substitution of a glutamic acid at position 44 with a proline, for example substitution of the amino acid at position 257 corresponding to valine with cysteine; Substitution of the amino acid at the position corresponding to paragine with histidine, and substitution of the amino acid at position 340 with glutamic acid and the substitution of the amino acid at position 232 with proline, A variant having a substitution of the amino acid at position α to lysine or arginine. (11)-(3)-(2)-(1)-(6) Substitution of glutamic acid at position 44, substitution of valine at position 257, substitution of asparagine at position 144, substitution of glutamic acid at position 340, and substitution of glutamine at position 249 Acid substitution, e.g., substitution of the amino acid at the position corresponding to glutamic acid at position 44 with proline , substitution of the amino acid at position 257 corresponding to valine with cysteine, and substitution of aspartate at position 262 Substitution of the amino acid at the position corresponding to lagin with histidine, corresponding to glutamic acid at position 340 substitution of the amino acid at position 249 with proline, and the amino acid at position 250 with glutamic acid A mutant having an amino acid substitution of lysine or arginine.

[0055] (11)-(4)-(2)-(1)-(6) Substitution of glutamic acid at position 44, substitution of glutamic acid at position 253, A substitution of valine at position 257, an asparagine at position 262, and a glutamic acid at position 340 For example, substitution of the amino acid at the position corresponding to glutamic acid at position 44 with proline; Substitution of the amino acid at the position corresponding to glutamic acid at position 253 with lysine or arginine; Substitution of the amino acid at the position corresponding to valine at position 257 with cysteine, The amino acid at position 340 corresponding to glutamic acid was replaced with histidine. A mutant having the amino acid at the corresponding position substituted with a proline. (11)-(8)-(4)-(2)-(1)-(6) Substitution of glutamic acid at position 44, substitution of glutamic acid at position 253 substitution, valine at position 257, asparagine at position 262, glutamic acid at position 340 and an aspartic acid at position 129, e.g., corresponding to a glutamic acid at position 44 substitution of the amino acid at position 253 corresponding to glutamic acid with proline; substitution of 257 amino acid with valine or lysine; Substitution of cysteine ​​into histidine at the amino acid position corresponding to asparagine at position 262 a substitution of the amino acid at the position corresponding to glutamic acid at position 340 with proline; and Substitution of the amino acid at the position corresponding to aspartic acid at position 129 with lysine or arginine A mutant having (11)-(10)-(4)-(2)-(1)-(6) Substitution of glutamic acid at position 44, substitution of glutamic acid at position 133 substitution of glutamic acid at position 253, substitution of valine at position 257, and asparagine at position 262 and a substitution of glutamic acid at position 340, e.g., corresponding to glutamic acid at position 44. substitution of the amino acid at position 133 with proline; to alanine, lysine at the amino acid position corresponding to glutamic acid at position 253 or Substitution with arginine, substitution with cysteine ​​at the amino acid position corresponding to valine at position 257 , a substitution of the amino acid at the position corresponding to asparagine at position 262 with histidine, and A mutant having a substitution of the amino acid at the position corresponding to the glutamic acid at position 14 with a proline. (11)-(10)-(4)-(2)-(1)-(5)-(6) Substitution of glutamic acid at position 44, glutamic acid at position 133 substitution, glutamic acid substitution at position 253, valine substitution at position 257, asparagus substitution at position 262 substitution of lysine for glutamine at position 337 and substitution of glutamic acid at position 340 For example, substitution of the amino acid at position 44 corresponding to glutamic acid with proline; Substitution of the amino acid at the position corresponding to glutamic acid at position 3 with alanine; Substitution of the amino acid at the position corresponding to lysine or arginine, and substitution of valine at position 257 Substitution of the amino acid at the corresponding position with cysteine, at the position corresponding to asparagine at position 262 substitution of the amino acid at position 337 with histidine; substitution with sine or arginine and the amino acid at the position corresponding to glutamic acid at position 340 A mutant having a substitution of α-aminopropyl to proline.

[0056] The amadoriase mutant of the present invention has excellent surfactant resistance and is The sequence may have amino acid substitutions that result in improved detergent resistance as described above. In addition, the detergent-resistant amadoriase mutant of the present invention has a structure in which the amino acid residues at positions other than the substituted amino acid residues are not present. and further one or several (for example, 1 to 15, for example, 1 to 10, preferably 1 to 5, More preferably, 1 to 3, and particularly preferably 1, amino acids are deleted, inserted, added, or Furthermore, the present invention provides the above-mentioned improved surfactant resistance. Amino acid substitution mutations that improve properties other than surfactant resistance, such as substrate specificity, and the substituted amino acid in the amino acid sequence shown in SEQ ID NO: 1 or 3. 70% or more, 75% or more, 80% or more of the amino acid sequence excluding non-acidic amino acids % or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and particularly Preferably, the nucleic acid has an amino acid sequence identity of 99% or more, amadoriase activity, and surface activity. The present invention also includes amadoriase mutants with altered drug resistance.

[0057] The amadoriase having the amino acid sequence shown in SEQ ID NO:1 was synthesized in accordance with the method described in WO 2006 / 023364. The recombinant plasmid designated pKK223-3-CFP-T7 in Conioc produced by E. coli carrying the FERM BP-10593 accession number The amadoriase (CFP-T7) derived from the Haeta genus is a thermostable enzyme previously discovered by the applicant. This CFP-T7 is a modified amadoriase with excellent quality. The amadoriase from the haeta genus had artificial nucleotides at positions 272, 302, and 388. This triple mutant was obtained by sequentially introducing multiple mutations.

[0058] In addition, SEQ ID NO: 3 is a mutant having improved substrate specificity as described in WO 2012 / 18094. (E98A) and International Publication No. 2007 / 125779 and International Publication No. 2013 / 10 The thermostability-improving mutations shown in No. 0006 (F43Y, G184D, carboxyl-terminal Amino acid residues of amadoriase from Coniochaeta sp. were deleted. It is an acid sequence.

[0059] In the above amino acid substitutions, the amino acid positions are the same as those shown in SEQ ID NO:1. The amino acid sequence of the amadoriase from the genus aeta is shown, but it may differ from that of other organisms. In the amino acid sequence of amadoriase derived from the species, the amino acid sequence shown in SEQ ID NO: 1 The amino acid at the position corresponding to the position in is replaced. More details will be given later.

[0060] In addition, as an amino acid substitution that improves the surfactant resistance, the amino acid shown in SEQ ID NO: 37 Substitutions of amino acids at positions corresponding to the following positions in the amino acid sequence are included: (i) substitution of glutamic acid at position 247, e.g., with lysine, arginine; (ii) substitution of glutamic acid at position 251, e.g., with lysine, arginine; (iii) substitution of threonine at position 335, e.g., with lysine or arginine; (iv) substitution of aspartic acid at position 230, e.g., with lysine or arginine; (v) substitution of aspartic acid at position 129, e.g., with lysine or arginine; (vi) substitution of aspartic acid at position 132, e.g., with lysine or arginine; (vii) Substitution of glutamic acid at position 133, e.g., alanine, methionine, lysine, arginine, Substitution for nin; (viii) substitution of asparagine at position 254, e.g., substitution with lysine or arginine; and (ix) Substitution of glutamic acid at position 229, e.g., with lysine or arginine.

[0061] The amadoriase mutants with improved detergent resistance have at least the above amino acid substitutions. It is sufficient to have one amino acid substitution, but it may also have multiple amino acid substitutions. It has 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions.

[0062] Among these, preferred are mutants having amino acid substitutions corresponding to the following amino acid positions: I wish. In the amino acid sequence shown in SEQ ID NO: 37, the following (i) to (iv): (i) Substitution of the amino acid at the position corresponding to glutamic acid at position 251 with lysine and a variant having a substitution of lysine for the amino acid at the position corresponding to threonine at position; (ii) Substitution of the amino acid at the position corresponding to aspartic acid at position 132 with lysine and a variant having a substitution of the amino acid at the position corresponding to threonine at position 5 with lysine; (iii) Substitution of the amino acid at the position corresponding to glutamic acid at position 133 with alanine and A variant having a substitution of the amino acid at the position corresponding to threonine at position 35 with lysine; and (iv) Substitution of the amino acid at the position corresponding to glutamic acid at position 229 with lysine and A mutant having a substitution of the amino acid at the position corresponding to the threonine at position 2 with lysine.

[0063] (Obtaining the gene encoding amadoriase) The genes of the present invention that code for these amadoriases (hereinafter, simply referred to as "amadoriase genes") To obtain a gene, the commonly used gene cloning method is used. For example, amadoriase can be produced by conventional methods from microbial cells or various cells capable of producing amadoriase. For example, Current Protocols in Molecular Biol The chromosome was then cloned by the method described in (Wiley Interscience, 1989). DNA or mRNA can be extracted. Furthermore, cDNA can be produced using mRNA as a template. Using the chromosomal DNA or cDNA thus obtained, Libraries of chromosomal DNA or cDNA can be made.

[0064] Next, an appropriate probe DNA was synthesized based on the amino acid sequence of the amadoriase. This was used to isolate amadoriase genes from chromosomal DNA or cDNA libraries. Alternatively, a suitable primer DNA is prepared based on the amino acid sequence. Then, perform an appropriate polymerase chain reaction (PCR) such as 5'RACE or 3'RACE. DNA containing the desired gene fragment encoding amadoriase is amplified by The DNA fragments are ligated to obtain DNA containing the full length of the desired amadoriase gene. can be done.

[0065] A preferred example of the gene encoding amadoriase thus obtained is C The amadoriase gene from the genus Oniochaeta (see Japanese Patent Publication No. 2003-235585) Examples of such cases include the use of the Internet for advertising purposes.

[0066] These amadoriase genes were ligated to various vectors in the usual manner. It is preferable for handling. For example, Coniochaeta sp. NISL 9330 strain The DNA encoding the amadoriase gene was inserted into pKK223-3 Vector (GE The recombinant plasmid pKK223-3-CFP (Patent Publication No. 2002-010011) was inserted into (Patent Publication No. 3-235585).

[0067] (vector) The vectors that can be used in the present invention are not limited to the above-mentioned plasmids. Any vector known to those skilled in the art, such as bacteriophage, cosmid, etc., may be used. Specifically, for example, pBluescriptII SK + (manufactured by STRATAGENE) is preferred.

[0068] (Mutation of the amadoriase gene) The mutation of the amadoriase gene can be carried out by any known method depending on the intended mutation form. That is, the amadoriase gene or a gene containing the gene can be A method of contacting and reacting recombinant DNA with a mutagenic agent; ultraviolet irradiation method; genetic engineering A wide range of techniques, including those utilizing biological techniques or protein engineering techniques, can be used.

[0069] Examples of mutagenic agents used in the above mutation treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, hydrazine, ants Examples of the uracil include 5-bromouracil and 5-bromouracil.

[0070] The above-mentioned contact and action conditions can be set according to the type of drug used, etc. However, there are no particular limitations as long as the desired mutation can be actually induced in the amadoriase gene. Generally, the above drug concentration is preferably 0.5 to 12 M, and the reaction temperature is 20 to 80°C. By contacting and reacting the mixture at a temperature for 10 minutes or more, preferably for 10 to 180 minutes, the desired When UV irradiation is performed, it should be performed according to the standard method as described above. (Modern Chemistry, pp. 24-30, June 1989 issue).

[0071] Generally, the methods that utilize protein engineering techniques are Site-Specific A technique known as c mutagenesis can be used. For example, Kra mer method (Nucleic Acids Res.,12,9441(1984):Me thods Enzymol.,154,350(1987):Gene,37,73( 1985)), Eckstein method (Nucleic Acids Res.,13,8 749(1985):Nucleic Acids Res.,13,8765(198 5): Nucleic Acids Res, 14, 9679 (1986)), Kunk el method (Proc. Natl. Acid. Sci. USA,82,488 (1985): Methods Enzymol., 154, 367 (1987) etc. Specific methods for converting the base sequence in DNA include, for example, (Transformer Mutagenesis Kit; Clonetech, EXOIII / Mung Bean Deletion Kit;Stratagen Made by e, Quick Change Site Directed Mutagenes Examples include the use of a ELISA Kit (e.g., Stratagene).

[0072] In addition, the general PCR method (polymerase chain reaction, Polymerase A technique known as the Chain Reaction can also be used (Tech In addition to the above genetic modification methods, organic synthesis and Alternatively, the desired modified amadoriase gene can be directly synthesized by enzymatic synthesis.

[0073] Determining or confirming the DNA base sequence of the amadoriase gene obtained by the above method In this case, for example, a multi-capillary DNA analysis system CEQ2000 (Beck This can be done using a spectrophotometer (manufactured by Mann Coulter) or the like.

[0074] (Transformation and transduction) The amadoriase gene obtained as described above is transformed into a bacteriophage, Vectors such as cosmids or plasmids used to transform prokaryotic or eukaryotic cells The vectors are then inserted into a host corresponding to each vector, and transformed or transduced in a conventional manner. For example, the resulting recombinant DNA can be used to transform any host, e.g. A microorganism belonging to the genus Escherichia, specifically, E. coli K-12 strain, preferably E. coli JM109 strain, E. coli DH5α strain (both manufactured by Takara Bio Inc.) or E. coli B strain, preferably Transform or transduce E. coli BL21 strain (Nippon Gene Co., Ltd.) and the like. Each strain can be obtained.

[0075] (Amino acid sequence identity) The identity of amino acid sequences was determined using the GENETYX Ver.11 (GeneTyx) matrix. Programs such as Maximum Matching and Search Homology or DNASIS Pro (Japanese Using programs such as maximum matching and multiple alignment (Ritsu Soft) It can be calculated.

[0076] (Identification of amino acid positions) The "position corresponding to the amino acid" refers to the amino acid sequence of the Coniochaeta amino acid sequence shown in SEQ ID NO:1. Amino acids at specific positions in the amino acid sequence of amadoriase from other organisms This refers to the position in the amino acid sequence of the dryase.

[0077] As a method for identifying the "position corresponding to an amino acid", for example, the Lippmann-Parson method The amino acid sequences of each amadoriase were compared using a known algorithm such as This can be done by giving maximum identity to conserved amino acid residues present in the alignment. By aligning the amino acid sequence of amadoriase in this manner, the amino acid sequence The sequence of each amadoriase sequence is determined by the homologous amino acid residues, regardless of the insertion or deletion within the sequence. Homologous positions are those that occur at the same position in the three-dimensional structure. These compounds are believed to have similar effects on the specific functions of the amadoriase of interest. It can be estimated.

[0078] 1-1, 1-2, and 1-3 show examples of amadoriase sequences derived from various known biological species. The amino acid sequence shown in SEQ ID NO: 1 is shown at the top. All of them have an identity of 70% or more with the sequence of SEQ ID NO: 1 and are aligned using a known algorithm. The mutation points in the mutants of the present invention are shown in the figures. Amino acids at specific positions in the amino acid sequence of amadoriase from the genus Coniochaeta. The position of the corresponding amino acid sequence of amadoriase from other organisms can be known. Figures 1-1, 1-2, and 1-3 show amadoriase derived from the genus Coniochaeta. (SEQ ID NO: 1), amadoriase from Eupenicillium terrenum (SEQ ID NO: 34), ketoamine oxidase from Pyrenochaeta sp. ( SEQ ID NO: 35), ketoamine oxidase from Arthrinium sp. (SEQ ID NO: No. 36), ketoamine oxidase from Curvularia clavata (sequence No. 37), ketoamine oxychloride from Neocosmospora vasinfecta sidase (SEQ ID NO: 38), from Cryptococcus neoformans Glucosyl amino acid oxidase (SEQ ID NO: 39), Phaeosphaeria no dorum (SEQ ID NO: 40), Fructosyl amino acid oxidase from Illus nidulans (SEQ ID NO: 41) , fructosyl amino acid oxidase from Ulocladium sp. (SEQ ID NO:4) 2) and fructosyl amino acids from Penicillium crysogenum The amino acid sequence of the oxidase (SEQ ID NO:43) is shown.

[0079] (Position corresponding to the replacement) In the present invention, "the amino acid sequence corresponding to glutamic acid at position 44 of the amino acid sequence shown in SEQ ID NO: 1" is The term "position corresponding to the amino acid sequence of the determined amadoriase" refers to the position corresponding to the amino acid sequence of the C When compared with the amino acid sequence of amadoriase from the Oniochaeta genus, sequence no. It means the amino acid corresponding to glutamic acid at position 44 of amadoriase No. 1. This allows the amino acid sequence to be identified by the above-mentioned method of identifying the "amino acid residues at the corresponding positions." This can be identified by the aligned Figure 1-1.

[0080] That is, in the case of amadoriase derived from Eupenicillium terrenum, Lysine at position 44, in ketoamine oxidase from Pyrenochaeta sp. Proline at position 44, ketoamine oxidase from Arthrinium sp. Proline at position 4, ketoamine oxidase from Curvularia clavata Proline at position 44, keto Proline at position 44 in amine oxidase; Cryptococcus neoformans In the ans-derived fructosyl amino acid oxidase, leucine at position 44 is present, whereas in the Phaeosp In the fructosyl peptide oxidase from Haeria nodorum, the 44th position of the protease Lorin, a fructosyl amino acid oxyester derived from Aspergillus nidulans proline at position 43 in the 5'-dimer enzyme, and fructosyl amino acid from Ulocladium sp. Proline at position 44 in acid oxidase; Penicillium crysogenum In the fructosyl amino acid oxidase derived from Escherichia coli, it is proline at position 44.

[0081] In addition, "the position corresponding to glutamic acid at position 81 of the amino acid sequence shown in SEQ ID NO: 1" means The determined amino acid sequence of amadoriase was determined from the Coniochaete When compared with the amino acid sequence of amadoriase from the genus ta, the amadoriase sequence of SEQ ID NO: 1 This also means the amino acid corresponding to glutamic acid at position 81 of ribozyme. The amino acid sequences can be identified from FIG. 1-1, which shows the alignment of the amino acid sequences by the method described above.

[0082] That is, in the case of amadoriase derived from Eupenicillium terrenum, Asparagine at position 81, ketoamine oxidase from Pyrenochaeta sp. glutamic acid at position 81 from zeta; ketoamine oxida from Arthrinium sp. histidine at position 81 in the enzyme, and ketoamine from Curvularia clavata In oxidase, glutamic acid at position 81 is found in Neocosmospora vasinfe Asparagine at position 81 in ketoamine oxidase from Cta; In the fructosyl amino acid oxidase from U.S. neoformans, the aspartate at position 81 is Paragine, a fructosyl peptide from Phaeosphaeria nodorum Glutamic acid at position 81 in oxidase, derived from Aspergillus nidulans In the fructosyl amino acid oxidase of In the fructosyl amino acid oxidase from Penicillin sp., glutamic acid at position 81 is 8 in the case of fructosyl amino acid oxidase from C. cillium chrysogenum The number one is asparagine.

[0083] In addition, "the position corresponding to glutamic acid at position 133 of the amino acid sequence shown in SEQ ID NO: 1" The determined amino acid sequence of amadoriase was compared with that of Coniocha, which is shown in SEQ ID NO:1. When compared with the amino acid sequence of amadoriase derived from the genus eta, the amino acid sequence of the amadoriase shown in SEQ ID NO: 1 This refers to the amino acid corresponding to glutamic acid at position 133 in the amino acid sequence of The amino acid sequences can be identified from FIG. 1-1, which shows the alignment of the amino acid sequences by the method described above.

[0084] That is, in the case of amadoriase derived from Eupenicillium terrenum, Glutamic acid at position 133, ketoamine oxida from Pyrenochaeta sp. The enzyme, glutamic acid at position 133, and the ketoamine oxyketone from Arthrinium sp. alanine at position 133 in the ketoacylase from Curvularia clavata In Neocosmospora vasi, glutamic acid at position 133 is In the ketoamine oxidase from Cryptococcus infecta, the alanine at position 133 is 133 position in fructosyl amino acid oxidase from C. neoformans glutamic acid from Phaeosphaeria nodorum, fructosyl peptide from Phaeosphaeria nodorum glutamic acid at position 131 in glutamate oxidase; Aspergillus nidula In the fructosyl amino acid oxidase derived from ns, glutamic acid at position 132 and Ulocl In the fructosyl amino acid oxidase from Adium sp., the lysine at position 133, P Fructosyl amino acid oxidase from Enicillium chrysogenum. In this case, it is aspartic acid at position 133.

[0085] In addition, "the position corresponding to glutamic acid at position 253 of the amino acid sequence shown in SEQ ID NO: 1" The determined amino acid sequence of amadoriase was compared with that of Coniocha, which is shown in SEQ ID NO:1. When compared with the amino acid sequence of amadoriase derived from the genus eta, the amino acid sequence of the amadoriase shown in SEQ ID NO: 1 This refers to the amino acid corresponding to glutamic acid at position 253 in the amino acid sequence of The amino acid sequences can be identified from FIG. 1-2, which shows the alignment of the amino acid sequences by the method described above.

[0086] That is, in the case of amadoriase derived from Eupenicillium terrenum, Alanine at position 253, ketoamine oxidase from Pyrenochaeta sp. In the case of alanine at position 251, ketoamine oxidase from Arthrinium sp. 253 glutamic acid, a ketoamine derived from Curvularia clavata In oxidase, glutamic acid at position 251 is found, whereas in Neocosmospora vasinf In the ketoamine oxidase from Cryptococcus ecta, valine is at position 253. In the fructosyl amino acid oxidase from S. neoformans, the 253 position of the glutamate is Tamic acid, a fructosyl peptide from Phaeosphaeria nodorum Arginine at position 249 in oxidase from Aspergillus nidulans In the fructosyl amino acid oxidase of In the fructosyl amino acid oxidase from Penicillin sp., glutamic acid was found at position 251. Fructosyl amino acid oxidase from C. cillium chrysogenum Ranked 53rd is glutamine.

[0087] Furthermore, "the position corresponding to glycine at position 256 of the amino acid sequence shown in SEQ ID NO: 1" means The determined amino acid sequence of amadoriase was determined from the Coniochaete When compared with the amino acid sequence of amadoriase from the genus ta, the amadoriase sequence of SEQ ID NO: 1 This refers to the amino acid corresponding to the glycine at position 256 of ribozyme. This can be identified from Figure 1-2, which shows an alignment of amino acid sequences.

[0088] That is, in the case of amadoriase derived from Eupenicillium terrenum, Asparagine at position 256, ketoamine oxida from Pyrenochaeta sp. Aspartic acid at position 254 in the enzyme, and ketoamine thiol derived from Arthrinium sp. In oxidase, glycine at position 256 is Asparagine at position 254 in amine oxidase, Neocosmospora vas infectioenza ketoamine oxidase, glycine at position 256; In the case of fructosyl amino acid oxidase from Occus neoformans, 256 Glutamic acid, fructosyl ester from Phaeosphaeria nodorum Asparagine at position 252 in Aspergillus nidul In the ans-derived fructosyl amino acid oxidase, asparagine at position 256 and Uloc In the case of fructosyl amino acid oxidase from ladium sp., asparagus at position 254 Gin, a fructosyl amino acid derived from Penicillium crysogenum In glycerylase, it is aspartic acid at position 256.

[0089] Furthermore, "a position corresponding to valine at position 257 of the amino acid sequence shown in SEQ ID NO: 1" means The determined amino acid sequence of amadoriase was determined by the Coniochaet When compared with the amino acid sequence of amadoriase derived from the amadoriase genus a, the amadoriase sequence of SEQ ID NO: 1 This refers to the amino acid corresponding to valine at position 257 of the ribozyme. It can be identified from the amino acid sequence alignment shown in FIG. 1-2.

[0090] That is, in the case of amadoriase derived from Eupenicillium terrenum, Valine at position 257, in ketoamine oxidase from Pyrenochaeta sp. is a threonine at position 255, and ketoamine oxidase from Arthrinium sp. The 257th cysteine, a ketoamine thiol derived from Curvularia clavata, Valine at position 255 in oxidase, Neocosmospora vasinfecta The ketoamine oxidase from Cryptococcus In the fructosyl amino acid oxidase from C. neoformans, the cysteine ​​at position 257 is fructosyl peptide oxidant derived from Phaeosphaeria nodorum Serine at position 253 in the enzyme, and fructosine from Aspergillus nidulans In the case of selenoamino acid oxidase, threonine at position 257 is from Ulocladium sp. In the previous fructosyl amino acid oxidase, valine at position 255 was found. In the fructosyl amino acid oxidase from C. crysogenum, valine at position 257 is It is.

[0091] Furthermore, "the position corresponding to asparagine at position 262 of the amino acid sequence shown in SEQ ID NO: 1" The amino acid sequence of the determined amadoriase is compared with that of the Conioch When compared with the amino acid sequence of amadoriase from the genus aeta, the amadoriase of SEQ ID NO: 1 This refers to the amino acid corresponding to asparagine at position 262 of the lyase. The amino acid sequences can be identified from FIG. 1-2, which shows the alignment of the amino acid sequences by the method described above.

[0092] That is, in the case of amadoriase derived from Eupenicillium terrenum, Aspartic acid at position 262, ketoamine oxy from Pyrenochaeta sp. Asparagine at position 260 in the ketoamine thiolase from Arthrinium sp. In the case of oxidase, histidine at position 262 was found in the case of ketoxidase derived from Curvularia clavata. Asparagine at position 260 in tocopherol oxidase; sinfecta ketoamine oxidase has a histidine at position 262; In the case of fructosyl amino acid oxidase from O. neoformans, Asparagine at position 62, fructose from Phaeosphaeria nodorum Asparagine at position 258 in Aspergillus nidulans peptide oxidase; In the fructosyl amino acid oxidase from ulans, aspartic acid at position 262 and U In the fructosyl amino acid oxidase from Loca- dium sp., the 260th amino acid is Sparagine, a fructosyl amino acid derived from Penicillium crysogenum In acid oxidase, it is aspartic acid at position 262.

[0093] Furthermore, "a position corresponding to glutamine at position 337 of the amino acid sequence shown in SEQ ID NO: 1" The determined amino acid sequence of amadoriase was compared with that of Coniocha, which is shown in SEQ ID NO:1. When compared with the amino acid sequence of amadoriase derived from the genus eta, the amadoriase of SEQ ID NO: 1 This refers to the amino acid corresponding to glutamine at position 337 of GlycA-ase. This can be identified from Figure 1-2, in which the amino acid sequences are aligned by the method.

[0094] That is, in the case of amadoriase derived from Eupenicillium terrenum, Lysine at position 337, in ketoamine oxidase from Pyrenochaeta sp. at lysine 335, whereas in ketoamine oxidase from Arthrinium sp. Glutamine at position 338, a ketoamine oxysaccharide derived from Curvularia clavata Threonine at position 335 in Neocosmospora vasinfecta The ketoamine oxidase from Cryptococcus ne In the fructosyl amino acid oxidase from P. obormans, the lysine at position 337 is Fructosyl peptide oxidase from Aeosphaeria nodorum Lysine at position 333, fructosyl amine from Aspergillus nidulans Asparagine at position 337 in oleic acid oxidase, and fluorouracil from Ulocladium sp. In glyceryl amino acid oxidase, threonine at position 335 is required. In the fructosyl amino acid oxidase from C. crysogenum, the lysine at position 337 be.

[0095] Furthermore, "the position corresponding to glutamic acid at position 340 in the amino acid sequence shown in SEQ ID NO: 1" The amino acid sequence of the determined amadoriase is compared with that of the Conioch When compared with the amino acid sequence of amadoriase from the genus aeta, the amadoriase of SEQ ID NO: 1 This refers to the amino acid corresponding to glutamic acid at position 340 of the lyase. The amino acid sequences can be identified from FIG. 1-2, which shows the alignment of the amino acid sequences by the method described above.

[0096] That is, in the case of amadoriase derived from Eupenicillium terrenum, Glutamic acid at position 340, ketoamine oxida from Pyrenochaeta sp. The enzyme, glutamic acid at position 338, and the ketoamine oxide from Arthrinium sp. In the case of glyceryl esterase, glutamic acid at position 341 is found in the glyceryl esterase from Curvularia clavata. In catabolic acid oxidase, glutamic acid at position 338 is required. sinfecta ketoamine oxidase, proline at position 340; 34 in fructosyl amino acid oxidase from Coccus neoformans Glutamic acid at position 0, fructosyl from Phaeosphaeria nodorum In peptide oxidase, lysine at position 336 is In the fructosyl amino acid oxidase derived from s, glutamic acid at position 340 and Ulocla In the case of fructosyl amino acid oxidase from D. tumefaciens, glutamic acid at position 338 is , fructosyl amino acid oxidant from Penicillium crysogenum In α-glutamic acid, it is the 340th glutamic acid.

[0097] Furthermore, "the position corresponding to aspartic acid at position 129 of the amino acid sequence shown in SEQ ID NO: 1" " refers to the amino acid sequence of the determined amadoriase, which is synthesized by the Conioc method shown in SEQ ID NO:1. When compared with the amino acid sequence of amadoriase from the genus Haeta, the amadoriase of SEQ ID NO: 1 This refers to the amino acid corresponding to aspartic acid at position 129 of the lyase. can also be identified from FIG. 1-1 in which amino acid sequences are aligned by the above method.

[0098] That is, in the case of amadoriase derived from Eupenicillium terrenum, Glutamic acid at position 129, ketoamine oxida from Pyrenochaeta sp. Aspartic acid at position 129 in the enzyme, and ketoamine thiol derived from Arthrinium sp. Aspartic acid at position 129 in oxidase, from Curvularia clavata In the ketoamine oxidase of Neocosmospora , aspartic acid is inserted at position 129. In the ketoamine oxidase from Vasinfecta, aspartic acid is at position 129. Fructosyl amino acid oxidants from Cryptococcus neoformans Serine at position 129 in the enzyme, fructooligosaccharide derived from Phaeosphaeria nodorum Aspartic acid at position 127 in tosylpeptide oxidase; In the fructosyl amino acid oxidase from C. nidulans, glutamic acid at position 128 , and 129th position in fructosyl amino acid oxidase from Ulocladium sp. aspartic acid from Penicillium crysogenum, and fructosyl from Penicillium crysogenum In amino acid oxidase, it is glutamic acid at position 129.

[0099] Furthermore, "the position corresponding to aspartic acid at position 132 of the amino acid sequence shown in SEQ ID NO: 1" " refers to the amino acid sequence of the determined amadoriase, which is synthesized by the Conioc method shown in SEQ ID NO:1. When compared with the amino acid sequence of amadoriase from the genus Haeta, the amadoriase of SEQ ID NO: 1 This refers to the amino acid corresponding to aspartic acid at position 132 of the lyase. can also be identified from FIG. 1-1 in which amino acid sequences are aligned by the above method.

[0100] That is, in the case of amadoriase derived from Eupenicillium terrenum, Aspartic acid at position 132, ketoamine oxy from Pyrenochaeta sp. Aspartic acid at position 132 in the ketoamine derivative from Arthrinium sp. Aspartic acid at position 132 in oxidase from Curvularia clavata In the previous ketoamine oxidase, aspartic acid at position 132 and Neocosmospor In the ketoamine oxidase from A. vasinfecta, glutamic acid at position 132 is Fructosyl amino acid oxidants from Cryptococcus neoformans Aspartic acid at position 132 in the enzyme, derived from Phaeosphaeria nodorum In the case of fructosyl peptide oxidase, aspartic acid at position 130 is In the fructosyl amino acid oxidase from Lus nidulans, the aspartate at position 131 is Paragic acid, fructosyl amino acid oxidase from Ulocladium sp. aspartic acid at position 132, and Penicillium crysogenum-derived In glyceryl amino acid oxidase, it is aspartic acid at position 132.

[0101] Furthermore, "the position corresponding to glutamic acid at position 231 of the amino acid sequence shown in SEQ ID NO: 1" The amino acid sequence of the determined amadoriase is compared with that of the Conioch When compared with the amino acid sequence of amadoriase from the genus aeta, the amadoriase of SEQ ID NO: 1 This refers to the amino acid corresponding to glutamic acid at position 231 of the lyase. The amino acid sequences can be identified from FIG. 1-2, which shows the alignment of the amino acid sequences by the method described above.

[0102] That is, in the case of amadoriase derived from Eupenicillium terrenum, Glutamic acid at position 231, ketoamine oxida from Pyrenochaeta sp. The enzyme, glutamic acid at position 229, and the ketoamine oxide from Arthrinium sp. In the case of ketosidase, glutamic acid at position 231 is found in the ketosidase from Curvularia clavata. In catabolic acid oxidase, glutamic acid is at position 229. In Neocosmospora valine, sinfecta ketoamine oxidase has a glutamic acid at position 231; Fructosyl amino acid oxidase from Tococcus neoformans Glutamic acid at position 231, fructosporum from Phaeosphaeria nodorum Histidine at position 227 in Aspergillus nidulans. In the fructosyl amino acid oxidase from Ulans, glutamic acid at position 231 and In the fructosyl amino acid oxidase from O. ocladium sp., the 229th position of the glutamate is thamine, a fructosyl amino acid derived from Penicillium crysogenum In xylidase, it is glutamic acid at position 231.

[0103] Furthermore, "the position corresponding to aspartic acid at position 232 of the amino acid sequence shown in SEQ ID NO: 1" " refers to the amino acid sequence of the determined amadoriase, which is synthesized by the Conioc method shown in SEQ ID NO:1. When compared with the amino acid sequence of amadoriase from the genus Haeta, the amadoriase of SEQ ID NO: 1 This refers to the amino acid corresponding to aspartic acid at position 232 of the lyase. can also be identified from FIG. 1-2 in which the amino acid sequences are aligned by the above method.

[0104] That is, in the case of amadoriase derived from Eupenicillium terrenum, Aspartic acid at position 232, ketoamine oxy from Pyrenochaeta sp. Aspartic acid at position 230 in the ketoamine from Arthrinium sp. Glutamic acid at position 232 in oxidase, from Curvularia clavata In the ketoamine oxidase of Neocosmospora , aspartic acid is inserted at position 230. vasinfecta ketoamine oxidase has glutamic acid at position 232, C Fructosyl amino acid oxidase from ryptococcus neoformans glycine at position 232 in the glycine family, and fructine from Phaeosphaeria nodorum. In tosylpeptide oxidase, glutamic acid at position 228 is required. In Aspergillus n In the fructosyl amino acid oxidase from idulans, glutamic acid at position 232 is In the fructosyl amino acid oxidase from Ulocladium sp., Aspartic acid, fructosyl acetate from Penicillium crysogenum In amino acid oxidase, it is aspartic acid at position 232.

[0105] Furthermore, "the position corresponding to glutamic acid at position 249 of the amino acid sequence shown in SEQ ID NO: 1" The amino acid sequence of the determined amadoriase is compared with that of the Conioch When compared with the amino acid sequence of amadoriase from the genus aeta, the amadoriase of SEQ ID NO: 1 This refers to the amino acid corresponding to glutamic acid at position 249 of the lyase. The amino acid sequences can be identified from FIG. 1-2, which shows the alignment of the amino acid sequences by the method described above.

[0106] That is, in the case of amadoriase derived from Eupenicillium terrenum, Lysine at position 249, in ketoamine oxidase from Pyrenochaeta sp. at lysine 247, whereas in ketoamine oxidase from Arthrinium sp. Histidine at position 249, ketoamine oxyester from Curvularia clavata In the case of Neocosmospora vasinfectase, glutamic acid at position 247 is In the ketoamine oxidase from Cryptococcus a, glutamic acid at position 249 is present. In the fructosyl amino acid oxidase from S. neoformans, the 249th position of the Tamic acid, a fructosyl peptide from Phaeosphaeria nodorum In oxidase, glutamic acid at position 245 is derived from Aspergillus nidulans. In the previous fructosyl amino acid oxidase, alanine at position 249 and Ulocladium In the fructosyl amino acid oxidase from Penici sp., the serine at position 247 is 249 in the fructosyl amino acid oxidase from Illium crysogenum It is the number one source of glutamine.

[0107] (Production of the amadoriase of the present invention) The bacteria thus obtained have the ability to produce amadoriase with excellent surfactant resistance. To produce the amadoriase using the strain, the strain is cultured by a conventional solid culture method. However, it is preferable to use a liquid culture method whenever possible.

[0108] The medium for culturing the above strains may contain, for example, yeast extract, tryptone, peptone, etc. , meat extract, corn steep liquor, or soybean or wheat bran infusion, etc. The nitrogen sources above are sodium chloride, potassium phosphate monobasic, potassium phosphate dibasic, and magnesium sulfate. Inorganic salts such as calcium, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate One or more types of sugar are added, and if necessary, carbohydrate raw materials, vitamins, etc. are appropriately added. can be.

[0109] The initial pH of the medium is suitably adjusted to pH 7 to 9.

[0110] Any conditions can be used for the culture, for example, a culture temperature of 20 to 42°C, Preferably, the incubation temperature is about 30° C. for 4 to 24 hours, and more preferably, the incubation temperature is about 30° C. The culture can be carried out at 40°C for 8 to 16 hours by submerged culture with aeration, shaking culture, static culture, etc. do.

[0111] After the cultivation is completed, amadoriase can be extracted from the culture by a conventional enzyme extraction method. For example, the cells can be subjected to ultrasonic destruction, grinding, etc., in a conventional manner, or Alternatively, the enzyme may be extracted using a lytic enzyme such as lysozyme, or in the presence of toluene, etc. The enzyme can be excreted from the cells by shaking or leaving at room temperature to cause lysis. The solution is then filtered, centrifuged, or the like to remove solids, and streptomycin is added as necessary. After removing nucleic acids using sulfate, protamine sulfate, manganese sulfate, etc., The mixture is fractionated by adding alcohol, acetone, etc., and the precipitate is collected to obtain the crude enzyme of amadoriase. get.

[0112] In order to obtain a purified amadoriase enzyme preparation from the above-mentioned crude amadoriase enzyme, for example, For example, gel filtration using Sephadex, Superdex, Ultrogel, etc.; Adsorption elution method using ion exchanger; electrophoresis using polyacrylamide gel, etc.; hydrolysis Adsorption and elution methods using hydroxyapatite; sucrose density gradient centrifugation and other sedimentation methods; affinity clotting Separation methods using molecular sieve membranes or hollow fiber membranes, etc. are appropriately selected. By carrying out these methods or a combination of these methods, a purified amadoriase enzyme preparation can be obtained. In this way, an amadoriase having the desired improved substrate specificity can be obtained. It is possible.

[0113] (Surfactant in the present invention) The surfactant in the present invention is an interface that enables the method for measuring HbA1c of the present invention. There are no particular limitations on the surfactant, and nonionic surfactants and ionic surfactants, for example, Examples of surfactants include cationic surfactants, anionic surfactants, and amphoteric surfactants. In the present specification, the surfactant is preferably a cationic surfactant or an anionic surfactant. When referring to an agent, unless otherwise specified, this expression is intended to include one or more surfactants. .

[0114] The surfactant in the present invention has a critical micelle concentration (CMC) of 70 mM or less, 50 mM or less. Bottom, 20mM or less, 10mM or less, 7mM or less, 6mM or less, 5mM or less, 4.5mM or less Bottom, 4mM or less, 3.5mM or less, 3mM or less, 2.5mM or less, 2mM or less, 1.5m In some embodiments, the present invention may be The critical micelle concentration of the clear surfactant may be 0.1 mM or 0.01 mM or higher. In most cases, the critical micelle concentration is 50 mM or less, more preferably 20 mM or less, and even more preferably 10 mM or less. Preferably, it is 10 mM or less, more preferably, it is 7 mM or less, more preferably, it is 6 mM or less. The critical micelle concentration is the concentration at which a surfactant is dissolved in solution. This is the critical concentration at which micelles are formed, and micelles are not formed at concentrations lower than this. Generally, the lower the critical micelle concentration, the more likely it is that a surfactant will form micelles even at low concentrations, and act as a surfactant. Those skilled in the art can easily obtain the desired surfactant by conventional techniques. The critical micelle concentration can be determined, e.g., by measuring the fluorescence of a fluorescent reagent that interacts with a surfactant. Use a commercially available kit that uses light changes to measure the critical micelle concentration of surfactants. (For example, PFP's Detergent Critical Micelle Concentration (CMC) Assay Kit, etc.)

[0115] For example, the CMC of octyltrimethylammonium bromide (C8, OTAB) is approximately 1 40 mM, and the CMC of decyltrimethylammonium chloride (C10) is approximately 65 mM. The CMC of decyltrimethylammonium bromide (C10) is approximately 70 mM. The CMC of dodecyltrimethylammonium chloride (C12) is approximately 20 mM. The CMC of dodecyltrimethylammonium bromide (C12, DTAB) is approximately 16 mM. The CMC of tetradecyltrimethylammonium chloride (C14, TTAC) is Approximately 4.5 mM, tetradecyltrimethylammonium bromide (C14, TTAB The CMC of hexadecyltrimethylammonium chloride (C16, The CMC of CTAC is about 1.3 mM, and that of hexadecyltrimethylammonium bromide is about 1.3 mM. The CMC of amide (C16) is approximately 1 mM, and octadecyltrimethylammonium chloride The CMC of octadecyltrimethylammonium chloride (C18, STAC) is approximately 0.3 mM. The CMC of nium bromide (C18, STAB) is approximately 0.3 mM (see, for example, J. P. HYS. COLLIDE. CHEM., 52, 130(1948), J. PH YS. CHEM., 66, 1839(1962), J. AM. OIL. CH EMISTS. SOC., 30, 74(1953), J. PHARM. SCI ., 54, 436(1965), KONINKI. NED. AKAD. WET EN. PROC. SER B, 58, 97(1955), J. PHYS. C HEM., 65, 1807(1961), J. AM. CHEM. SOC., 65, 692(1943), J. AM. CHEM. SOC., 69, 209 5(1947), J. COLLIDE. INTERFACE. SCI., 22, 430(1966), J. AM. CHEM. SOC., 70, 3803(1 948). The number of carbon chains of the longest substituent is shown in parentheses.

[0116] For example, the CMC of 1-dodecylpyridinium bromide (C12) is approximately 12 mM. The CMC of 1-dodecylpyridinium chloride (C12, 1-DPC) is approximately 14 mM. and the CMC of 1-tetradecylpyridinium bromide (C14) is approximately 2.9 mM. The CMC of 1-hexadecylpyridinium chloride (C16, 1-CPC) is approximately 0.6. mM, and the CMC of 1-hexadecylpyridinium bromide (C16, 1-CPB) is Approximately 0.9 mM, N-cetyl-4-methylpyridinium chloride (C16, 4Me- The CMC of 1-CPC is approximately 1.9 mM, and that of 1-octadecylpyridinium bromide is approximately 1.9 mM. The CMC is about 0.6 mM, and the CMC of 1-octadecylpyridinium chloride is about 0. 24 mM (see, e.g., J. COLLIDE. INTERFACE. SCI., 21, 522(1966), J. PHARM. SCI., 54, 436(1 965), TRANS. FARADAY. SOC., 62, 3244 (1966 ), J. AM. CHEM. SOC., 70, 3803(1948), REV. CHIM. AC. REP. POP. ROUM., 6, 309(1961) , J. AM. CHEM. SOC., 70, 3049 (1948). ).

[0117] The CMC of benzyldodecyldimethylammonium chloride is about 2.8 mM. The CMC of benzyltetradecyldimethylammonium chloride (C14, BDTAC) is Approximately 0.37 mM, benzyl cetyl dimethyl ammonium chloride (C16, BCD The CMC of AC is about 0.042 mM (see, for example, Surfactant Handbook, vol. 131 (196 0), J. COLLIDE. INTERFACE. SCI., 22, 430( (1966), J. COLLIDE.SCI., 8, 385 (1953)).

[0118] Examples of nonionic surfactants include polyoxyethylene alkyl ethers and fatty acids. Sorbitan esters, alkyl polyglucosides, fatty acid diethanolamides, alkyl molybdates, Noglyceryl ether and the like.

[0119] Examples of cationic surfactants include alkyl trimethyl ammonium salts, dialkyl trimethyl ammonium salts, and the like. Kyldimethylammonium salts, alkylbenzyldimethylammonium salts, pyridinium Salts, such as alkylpyridinium salts, phosphonium salts, such as alkylphosphonium salts; Imidazolium salts, for example alkylimidazolium salts, isoquinonium salts, for example alkyl phenylisoquinonium salts.

[0120] The cationic surfactant of the present invention is a quaternary ammonium salt represented by the following general formula: Examples of suitable cations include ammonium salts (I), pyridinium salts (II) and phosphonium salts (III). [ka] [In the formula, R 1 ~R 4 may be the same or different, and may be substituted or unsubstituted C1 to C2 0 represents alkyl, alkenyl, aryl or benzyl; Z - represents a monovalent anion .] [ka] [In the formula, R 5 is a substituted or unsubstituted C1-C 20 Each Ra is the same or may be different, and each represents a hydrogen atom or a substituted or unsubstituted C1-C 20 Alkyl, ar represents an aryl, aryl or benzyl; n represents an integer of 1 to 5; Z - is a monovalent anion ] [ka] [In the formula, R 6 ~R 9 may be the same or different, and may be substituted or unsubstituted C1 to C2 0 represents alkyl, alkenyl, aryl or benzyl; Z - represents a monovalent anion .]

[0121] Examples of quaternary ammonium salts include octyltrimethylammonium chloride ( OTAC), Octyltrimethylammonium bromide (OTAB), Decyltrimethyl Ammonium chloride, decyltrimethylammonium bromide (DTAB), dodecyl Trimethylammonium chloride, dodecyltrimethylammonium bromide, tetradecyl Tetradecyltrimethylammonium chloride (TTAC), Tetradecyltrimethylammonium tetrabromide (TTAB), hexadecyltrimethylammonium chloride (CTAC), Hexadecyltrimethylammonium bromide, Octadecyltrimethylammonium chloride Bromide, Octadecyltrimethylammonium bromide (STAB), Eicosyltrimethylammonium bromide Eicosyltrimethylammonium chloride, Eicosyltrimethylammonium bromide, Benzyldodecyl Benzyldimethyiammonium chloride, benzyldodecyldimethylammonium bromide ( BDDAB), benzyltetradecyldimethylammonium chloride (BDTAC), Benzyltetradecyldimethylammonium bromide, benzylcetyldimethylammonium Muchloride (BCDAC), Benzylcetyldimethylammonium bromide, Dioctyl Dimethylammonium chloride, and dioctyldimethylammonium bromide. Can be obtained.

[0122] Examples of pyridinium salts include 1-decylpyridinium chloride, 1-decylpyridinium chloride, 1-Dodecylpyridinium bromide, 1-dodecylpyridinium chloride (1-DPC), 1-dodecylpyridinium pyridinium bromide, 1-tetradecylpyridinium chloride, 1-tetradecylpyridinium 1-Hexadecylpyridinium bromide (1-CPC), 1-hexadecylpyridinium chloride (1-CPC) Cetylpyridinium bromide (1-CPB), N-cetyl-2-methylpyridinium chloride N-cetyl-3-methylpyridinium chloride, N-cetyl-4-methylpyridinium 1-Octadecylpyridinium chloride (4Me-1-CPC), 1-Octadecylpyridinium chloride, 1-Octadecylpyridinium chloride Tadecylpyridinium bromide, 1-eicosylpyridinium chloride and 1-eicosyl and pyridinium bromide.

[0123] Examples of the phosphonium salt include tetraethylphosphonium chloride and tetraethyl Phosphonium bromide, tributylmethylphosphonium chloride, tributylmethylphos phosphonium bromide, tributylmethylphosphonium iodide, tetrabutylphosphonium Chloride, Tetrabutylphosphonium bromide, Tetra-n-octylphosphonium chloride tetra-n-octylphosphonium bromide, tributyldodecylphosphonium chloride Tributyldodecylphosphonium bromide, tributylhexadecylphosphonium , tributylhexadecylphosphonium bromide (TBCPB), methyltri Phenylphosphonium chloride, methyltriphenylphosphonium bromide, methyltri Phenylphosphonium iodide, tetraphenylphosphonium chloride, tetraphenyl Examples include phosphonium bromides.

[0124] The anion Z that is the counterion of the cationic surfactant - For example, Cl - , Br - , I - etc.

[0125] Examples of anionic surfactants include linear alkylbenzene sulfonates, alkoxysilanes, and the like. Kyl sulfate, alpha-olefin sulfonate, polyoxyethylene alkyl ether Examples include sulfates, α-sulfofatty acid ester salts, and alkali metal salts of natural fatty acids. Such surfactants include, for example, sodium dodecyl sulfate (SDS). .

[0126] Examples of amphoteric surfactants include alkyl dimethylamine oxide, alkyl carbo xybetaine, etc.

[0127] (Kit containing the amadoriase of the present invention and a surfactant) The present invention provides a kit for measuring glycated hemoglobin, which contains amadoriase and a surfactant. The surfactant may be a non-ionic or ionic surfactant. The lyase and the detergent may be included in the kit as the same or as separate components. When the amadoriase and the surfactant are contained in the kit as the same component, the surfactant It is preferable that the amount of amadoriase is not inactivated by the amadoriase. When the kit contains a lyase and a surfactant, the surfactant should be at the final concentration at the time of measurement. A stock solution with a higher concentration may be used. Dilute this stock solution appropriately for the measurement. Prepare the working solutions.

[0128] The kit comprising the amadoriase and surfactant of the present invention further comprises a reagent for measuring αFVH, Proteases or peptidases for excising αFVH, other known stabilizers and buffers, The technology used in various kits for measuring αFVH is The present invention can be suitably used for the production of a kit containing the above-mentioned amadoriase and a surfactant. That is, the present invention relates to a method for the preparation of an amadoriase, comprising the steps of providing a suitable amadoriase and a surfactant. The present invention provides a method for producing a kit comprising a lyase and a surfactant, The surfactant and the surfactant may be provided as the same or separate components. When amadoriase and detergent are provided as part of the assay, they are administered immediately before the αFVH assay. can be mixed into

[0129] The amadoriase contained in the kit of the present invention is preferably a detergent contained in the kit. The residual activity (%) 5 minutes after adding the final concentration of In comparison, it is preferably 13% or more, more preferably 15% or more, and most preferably 19% or more. For example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more , 80% or more, 90% or more, 95% or more, or 99% or more. will be explained.

[0130] The amadoriase contained in the kit of the present invention is preferably at a final concentration at the time of measurement. , 110 μg / ml or less per 0.01% (w / v) of surfactant, e.g. 100 μg / m 1 or less, 70 μg / ml or less, or 50 μg / ml or less. The final concentration of the surfactant at the time of measurement should be 0.01% (w / v) or more, for example 0.0 2% (w / v) or more, 0.04% (w / v) or more, 0.05% (w / v) or more, 0.06 %(w / v) or more, 0.07%(w / v) or more, 0.08%(w / v) or more, 0.09% (w / v) or more, 0.1%(w / v) or more, 0.15%(w / v) or more, 0.2%(w / v) or more, 0.25% (w / v) or more, or 0.3% (w / v) or more. The final concentration at the fixed time is the concentration at which the component is finally diluted and glycosylated hemoglobin is measured. Therefore, the stock solution in the kit is higher in concentration than the final concentration at the time of measurement. may also be used.

[0131] The amadoriase contained in the kit of the present invention is represented by SEQ ID NO: 1 or SEQ ID NO: 37. Amadoriase having an amino acid sequence or an amadoriase having improved surfactant resistance, The variant may be, for example, a variant having a similar sequence to SEQ ID NO: 1 or SEQ ID NO: 37, for example, 70% or more. above, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, or having an amino acid sequence with 99% or more sequence identity, or having SEQ ID NO: 1 or SEQ ID NO: In the amino acid sequence of No. 37, one or several amino acids are modified, mutated, or deleted. The amino acid sequence may have deletions, substitutions, additions and / or insertions.

[0132] The amadoriase contained in the kit of the present invention is derived from Eupenicillium genus, P Genus yrenochaeta, Genus Arthrinium, Genus Curvularia, Neo cosmospora spp., Cryptococcus spp., Phaeosphaeria spp. , Aspergillus sp., Emericella sp., Ulocladium sp., Pe Genus nicillium, Genus Fusarium, Genus Achaetomiella, Acha genus etomium, genus Thielavia, genus Chaetomium, Gelasino spora spp., Microascus spp., Leptosphaeria spp., Ophiob olus sp., Pleospora sp., Coniochaetidium sp., Pichia Genus, Corynebacterium, Agrobacterium, Arthro The amadoriase may be a naturally occurring amadoriase derived from the genus Bacter or a mutant thereof. Such variants may optionally comprise an aspartate residue at position 262 of the amino acid sequence shown in SEQ ID NO: 1 or 3. Lagin, valine at position 257, glutamic acid at position 253, glutamine at position 337, and 340 glutamic acid at position 133, glutamic acid at position 44, glycine at position 256 , glutamic acid at position 81, aspartic acid at position 129, aspartic acid at position 132, 23 From the group consisting of glutamic acid at position 1, aspartic acid at position 232, and glutamic acid at position 249 The polypeptide may have one or more amino acid substitutions at positions corresponding to amino acids selected from the group consisting of If a manufacturer is a manufacturer, for example, a certain amadoriase can be identified by the test method described below or the evaluation method of Example 7. or its variant can be used in the kit of the present invention, i.e., has the desired surfactant resistance can be easily checked.

[0133] (Buffer) The kit or composition of the present invention contains a medicament containing a medicament having a pH of 5 or less, which is within the range where amadoriase activity is not inactivated. A buffer having a buffering capacity in the range of pH 6.0 to pH 10.0, preferably pH 6.0 to pH 8.0. Alternatively, a buffer solution may be added as appropriate. In this specification, the term "buffer" refers to a buffer solution unless otherwise specified. The term is intended to include one or more buffering agents. A buffer is a substance that maintains the pH of a solution within a certain range. A buffer is a solution that has a buffering effect (buffer capacity), and a buffer agent is a substance that gives a buffering effect to a solution. A buffer is composed of a weak acid and its salt. For example, if a buffer consists of phosphoric acid and its potassium salt, Since the base compound is phosphoric acid, it is convenient to call it a phosphate buffer. In addition, the concentration of a certain buffer is determined based on the concentration of the compound in its free form. This refers to the concentration of the base compound, including its conjugate salt form. For example, 100 mM Li When we say phosphate buffer, we mean the final concentration of phosphoric acid and its conjugate salts (e.g. This means that the total phosphate concentration, including all salts of phosphate (e.g. potassium phosphate), is 100 mM.

[0134] Among various buffers, the remaining activity of amadoriase in the presence of surfactants is particularly important. It is preferable that the enzyme maintains the activity or reduces the decrease in the residual activity. Such preferred buffers are particularly those having an amadoriase stabilizing effect or those according to the present invention. For example, HEPES is used at 500 mM (pH 7.0) and is It does not exhibit amadoriase stabilizing effect on amadoriase (CFP-T7, SEQ ID NO: 1). Therefore, HEPES does not fall under the category of a buffer having an amadoriase stabilizing effect of the present invention. As such, not all buffers have the ability to stabilize amadoriase. The buffer having an amadoriase stabilizing effect of the present invention not only keeps the pH of the solution constant, but also In addition, the buffer of the present invention has an effect of stabilizing amadoriase when buffering pH. The amadoriase stabilizing effect of the agent is the stabilization of amadoriase in the presence of a surfactant. This refers to the action of maintaining the residual activity or reducing the decrease in the residual activity. The enzyme stabilizing effect was observed in the presence of a surfactant in a solution without added buffer or in the solution of the present invention. Residual amadoriase activity in a solution using a buffer that does not have an amadoriase stabilizing effect, The residual amadoriase activity of the solution containing the buffer of the present invention can be evaluated by comparing it with that of the solution containing the buffer of the present invention. do.

[0135] Examples of buffers (buffer solutions) that can be used in the kit (composition) of the present invention include: Boric acid and / or its salt-containing borate buffer, Tris-HCl buffer, phosphoric acid and / or its salt Phosphate buffers containing salts, such as potassium phosphate buffers or sodium phosphate buffers, organic Acid buffers and / or organic acid buffers including salts thereof, such as tricarboxylic acid buffers and / or Tricarboxylic acid buffers including their salts, e.g., citric acid buffers including citric acid and / or its salts Buffers, including monocarboxylic acid buffers and / or salts thereof, such as acetic acid Examples of the buffer include acetate buffers and / or their salts. ACES (N-(2-acetamido)-2-aminoethyl ether) is a buffer that can be used in ethanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ), Bicin (N,N-bis(2-hydroxyethyl)glycine), Bis-Tris (Bis(2 -Hydroxyethyl)iminotris(hydroxymethyl)methane), CHES(N-cyclohexane EPPS (4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfone acid), HEPPSO (N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonyl acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), propanesulfonic acid), MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), POPSO (piperazine-1 ,4-bis(2-hydroxypropanesulfonic acid)), TAPS (N-tris(hydroxymethyl) Methyl-3-aminopropanesulfonic acid), TAPSO(3-[N-tris(hydroxymethyl)methyl TES (N-tris(hydroxymethyl)amino)-2-hydroxypropanesulfonic acid Methyl-2-aminoethanesulfonic acid), Tricine (N-tris(hydroxymethyl)methylglucan lysine) and / or salts thereof. ) [ka] [wherein n may be 0, 1, 2 or 3; R 10 are each independently H, OH, -CH2OH or Also included are buffers containing the compound of the formula (III) (which may be -COOH) and / or a salt thereof. phthalic acid buffers containing maleic acid and / or its salts, and maleic acid buffers containing maleic acid and / or its salts. fumaric acid buffers containing fumaric acid and / or its salts, glutaric acid and / or its salts and citraconic acid buffers containing citraconic acid and / or salts thereof. a mesaconic acid buffer containing mesaconic acid and / or a salt thereof; Malonic acid buffers containing tartaric acid and / or its salts, tartaric acid buffers containing succinic acid and / or its salts adipic acid buffer containing adipic acid and / or its salt; phosphorus Also included are dicarboxylic acid-based buffers such as malic acid buffers containing malic acid and / or its salts. With the exception of HEPES and CHES, these are the amadoriases of the present invention. The buffer having a stabilizing effect of the present invention may be a buffer having a stabilizing effect. The buffers used are phosphate buffer, ACES buffer, citrate buffer, malic acid buffer, Acetic acid buffer, maleic acid buffer, citraconic acid buffer, malonic acid buffer, glutaric acid buffer buffers represented by formula (IV), such as MES buffer, MOPS buffer These include, but are not limited to, the following buffers: In addition, the amadoriase stabilizing effect of the present invention can be achieved by using one or more of the above-mentioned enzymes. The buffer having the above-mentioned function may be replaced with a substance other than the above-mentioned buffer (e.g., a buffer having no amadoriase stabilizing function). The salts may be sodium salts or potassium salts of the base compound. , magnesium salts, calcium salts and ammonium salts.

[0136] The buffers of the present invention can be used in the kits or compositions of the present invention at any suitable concentration. Generally, the amount of the buffer of the present invention added to the kit or composition of the present invention is set to be equal to the final concentration in the measurement solution. In one embodiment, the mitochondrial activity of the present invention in the measurement solution can be calculated based on the mitochondrial activity. The final concentration of the buffer is preferably sufficient to buffer any pH changes that may occur in the test solution. In another embodiment, the final concentration of the buffer of the present invention in the measurement solution is The residual activity of amadoriase in a solution containing the compound is 20% or more, preferably 40% or more, and more preferably The final concentration of the buffer of the present invention is preferably 60% or more, and more preferably 80% or more. For example, 1 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, for example, 50 mM or more, 1 M or less, 500 mM or less, 400 mM or less, 300 mM or less, 200 mM or less, 100 mM or less, for example, 1 mM to 1 M, 5 mM to 500 mM, 10 mM to 300 mM, The buffer having an amadoriase stabilizing effect of the present invention may be, for example, 50 mM to 100 mM. When a phosphate buffer is used, its concentration is 50 mM to 500 mM, for example, 50 mM to 300 mM, preferably The buffer of the present invention may be a citrate buffer, a malic acid buffer, a malic acid buffer, or the like. acid, citraconic acid buffer, malonic acid buffer, glutaric acid buffer, or tartrate buffer When used, the concentration is 5 mM to 500 mM, preferably 10 mM to 200 mM, for example, 10 mM to 100 mM. As the buffer of the present invention, a buffer represented by formula (IV), for example, an MES buffer, an MOPS buffer, When a buffer or a MOPSO buffer is used, the concentration is 10 mM to 500 mM, for example 100 mM to 500 mM, for example For example, the concentration may be 150 mM to 300 mM. When an ACES buffer is used as the buffer of the present invention, the concentration The buffer concentration may be 200 mM to 1 M, for example, 200 mM to 500 mM. The amount of the buffer used in the composition of the present invention may vary depending on whether or not the composition also contains a stabilizer. If added, the amount of the stabilizer may vary.

[0137] (Stabilizer) The kit or composition of the present invention contains a compound capable of maintaining the residual activity of amadoriase in the presence of a surfactant. A stabilizer may be added as appropriate to maintain the activity of the enzyme or to reduce the decrease in the residual activity. The stabilizer is a substance that maintains the residual activity of amadoriase in the presence of a surfactant or reduces the residual activity. In this specification, unless otherwise specified, the term "stabilizer" refers to a substance that reduces the decrease in This expression is intended to encompass one or more stabilizing agents. Examples of stabilizers that can be used include phosphoric acid, tricarboxylic acids (e.g., citric acid), , dicarboxylic acids (e.g., malic acid, maleic acid, citraconic acid, malonic acid, glutaric acid , tartaric acid), monocarboxylic acids (e.g., acetic acid), compounds represented by formula (IV) (e.g., ME S, MOPS, MOPSO), ammonium sulfate, their salts and any combination thereof Examples include:

[0138] The stabilizer of the present invention can be used in the kit or composition of the present invention at any suitable concentration. Generally, the amount of stabilizer added to the kit or composition of the present invention is set to the final concentration in the measurement solution. In one embodiment, the amount of stabilizer added is calculated based on the amount of surfactant. The residual activity of amadoriase in the solution is 35% or more, 37.5% or more, preferably 40% or more, 45% or more. or more, 50% or more, 55% or more, preferably 60% or more, 65% or more, 70% or more, 75% or more, Alternatively, the amount is 80% or more, 85% or more, 90% or more, or 95% or more. For example, the final concentration of the measurement solution is 0.1 mM to 100 mM, 0.2 mM to 100 mM, 0.5 mM to 50 mM, 1 mM to 30 mM, The compound may be added to the kit or composition so as to give a concentration of 10 mM, 2 mM to 30 mM, 5 mM to 20 mM, or 10 mM to 20 mM. If a buffer is also added to the composition of the invention, the amount of stabilizer may vary depending on the amount of the buffer. For example, when a stabilizer is added, the type and amount of buffer added to prevent a change in pH is important. The amount of the stabilizer may be appropriately selected and adjusted, and the pH of the stabilizer solution may be appropriately adjusted.

[0139] Among the buffers of the present invention, the phosphate buffer, the citrate buffer, and the MES buffer are particularly For example, the concentration of a phosphate buffer is the concentration at which the buffer capacity to maintain the pH of the solution constant is maintained. For example, 100 mM, for example, for citrate buffer, 50 mM, for example, for MES buffer, 150 mM. However, when used as a pH buffer, it was found to have an amadoriase stabilizing effect. While keeping the pH of the solution within a certain range using HEPES, which does not have an amadoriase stabilizing effect, Phosphoric acid and / or its potassium salt, citric acid and / or its sodium salt to be added to the composition Alternatively, even if the concentration of MES and / or its sodium salt is further reduced, the surfactant is not present. The residual activity of amadoriase was stabilized in the presence of phosphate and and / or its potassium salt, citric acid and / or its sodium salt, and MES and / or The sodium salt is at a lower concentration than the concentration at which it effectively exerts its buffering effect, specifically, at phosphoric acid. This was also observed at 5 mM for selenium, 0.5 mM for citrate, and 20 mM for MES. phosphoric acid and / or its potassium salt, citric acid and / or its sodium salt, Regarding MES and / or its sodium salt, the amadoriase as a buffer of the present invention is In addition to the stabilizing effect, it was confirmed that there is another stabilizing effect that allows amadoriase activity to remain. For the sake of convenience, in order to distinguish from the amadoriase stabilizing effect of the buffer of the present invention, Such an effect may be referred to as the amadoriase stabilizing effect of the stabilizer of the present invention. Phosphoric acid and / or its potassium salt, citric acid and / or its sodium salt, and MES and and / or its sodium salt have the amadoriase stabilizing effect of the buffer of the present invention, and The stabilizer of the present invention has an amadoriase stabilizing effect. In other words, phosphate and and / or its potassium salt, citric acid and / or its sodium salt, and MES and / or its The sodium salt of the above corresponds to the buffering agent of the present invention, and also corresponds to the stabilizer of the present invention.

[0140] In addition, maleic acid, citraconic acid, malonic acid, and glutamic acid, which have amadoriase stabilizing properties, Tartaric acid, tartaric acid, MOPS, and MOPSO are buffered at concentrations lower than their effective buffering effect. Amadoriase stabilization was observed at 10 mM or 20 mM, but these were not observed at higher concentrations ( 50 mM, 100 mM, 150 mM, etc.) is a compound that can exert a buffering effect. , citraconic acid, malonic acid, glutaric acid, tartaric acid, MOPS, MOPSO, and the compound represented by formula (IV) The compound also falls within the scope of the buffer of the present invention, and also falls within the scope of the stabilizer of the present invention.

[0141] The kit or composition of the present invention comprises an amadoriase, a surfactant, a stabilizer and / or a buffer. When buffers are included, they can be added to the kit or composition in any order. The kit or composition of the present invention may further comprise a stabilizer and / or a buffer (if included). Adding the surfactant after the addition of the α-amadoriase reduces the residual activity of the amadoriase. can be reduced.

[0142] (Improvement of Surfactant Resistance in the Amadoriase of the Present Invention) The amadoriase of the present invention obtained by the above-mentioned method can be modified by gene modification or the like. As a result of the mutation in the amino acid sequence, the surfactant resistance is improved compared to the unmodified version. Specifically, the activity of the modified amadoriase is higher than that of the unmodified amadoriase. The lyase was selected from the group consisting of the lyases described in the activity measurement method and the surfactant resistance evaluation method described in this specification. Under reaction conditions, a given surfactant treatment, for example, 0.01% (w / v) hexadecyltrate After adding trimethylammonium chloride (hereinafter referred to as "CTAC"), The remaining activity (%) after 5 minutes is improved. The percentage of activity after surfactant treatment is calculated based on the activity before surfactant treatment taken as 100. In this specification, the concentration of the surfactant is expressed as a percentage (%). When percentages are stated, this means % (w / v) unless otherwise stated. .

[0143] The degree of improvement in the residual activity (%) of the modified amadoriase of the present invention is not limited, but may be, for example, Amadoriase before and after the introduction of the mutation of the present invention was treated with a detergent. The residual activity (%) is preferably 13% or more, more preferably 15% or more, and most preferably 1 9% or more, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 7 Modified amadorias that are 0% or more, 80% or more, 90% or more, 95% or more, or 99% or more The present invention encompasses amadoriases before and after the introduction of the present mutations. In the numerical comparison of the residual activity (%) after surfactant treatment, it is preferable that the residual activity is 2% or more. or 9% or more, most preferably 19% or more, for example 20% or more, 30% or more, 40% or more Above, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or The present invention encompasses modified amadoriases whose residual activity is improved by 9% or more.

[0144] In one embodiment, the amadoriase prior to introduction of the mutations of the present invention is treated with a detergent. In such cases, the activity may be completely lost after the introduction of the mutation of the present invention. In order to see an improvement in the residual activity (%) of amadoriase, the activity was also increased by the surfactant treatment. Using a standard amadoriase that does not disappear completely, the interfacial activity of the standard amadoriase was measured. The residual activity of the amadoriase after treatment with a detergent and the residual activity of the amadoriase after the mutation introduction were measured. Comparisons may be made.

[0145] In fact, in addition to the temperature conditions during measurement, the detergent resistance of amadoriase before the introduction of mutations was also important. The relative evaluation results will differ depending on the degree of residual activity (%) and residual activity ratio. It is difficult to evaluate the absolute detergent resistance of each mutant by only comparing the magnitude of the values. However, by following the conditions of the examples of the present invention, the surfactant resistance of each mutant can be improved. This allows for absolute evaluation, and facilitates the selection of the amadoriase of the present invention. The residual activity (%) of amadoriase before the mutation introduction is calculated to be sufficiently low. By selecting the treatment conditions, the degree of improvement in the residual activity (%) and the residual activity can be generally improved. There is a tendency for the sex ratio to be calculated high.

[0146] For example, the E. coli JM109 (pKK223-3-CFP-T7 / The amadoriase of the present invention produced by the strain 253K was mixed with 0.01% CTAC, When subjected to 30° C. for 5 minutes, the amadoriase C The residual activity of FP-T7 was 69.9%, whereas the residual activity of Amadoriar after the introduction of the mutations of the present invention was 10.1%. The enzyme had a residual activity of more than 72%. The amadoriase of the present invention produced by the strain D7) was mixed with 0.04% CTAC and incubated for 30 When the enzyme was subjected to heating at 5° C. for 5 minutes, the amadoriase CFP before the introduction of the mutation of the present invention was The residual activity of the amadoriase after the introduction of the mutations of the present invention was 12.7%. The residual activity of the amadoriase with improved surfactant resistance is more than 15%. The shelf life of products containing glycerol is significantly improved, and the protease decomposition efficiency of HbA1c is also improved. and is stable even when strong surfactants are used to increase sensitivity. This is extremely advantageous industrially.

[0147] (Method for measuring amadoriase activity) Various methods can be used to measure the activity of amadoriase. The method for measuring amadoriase activity used in the present invention will now be described.

[0148] (Method for measuring amadoriase activity) In the present invention, the enzymatic activity of amadoriase can be measured by measuring the amount of amadoriase produced by the enzyme reaction. There are two methods: one to measure the amount of hydrogen peroxide produced and the other to measure the amount of oxygen consumed by an enzyme reaction. The main measurement methods are as follows. As an example, the method for measuring the amount of hydrogen peroxide is as follows. Shown below.

[0149] In the following description, unless otherwise specified, the amadoriase activity measurement in the present invention is carried out using fructosyl Valine is used as a substrate. The enzyme activity is measured using fructosyl valine as a substrate. The amount of enzyme that produces 1 μmol of hydrogen peroxide per minute when fructosporin is activated is defined as 1 U. Glycated amino acids such as silverline and glycated peptides such as fructosyl valyl histidine It can be synthesized and purified according to the method of Sakagami et al. (see JP-A-2001-95598). ).

[0150] A. Preparation of Reagents (1) Reagent 1: POD-4-AA solution 4.0 kU of peroxidase (Kikkoman), 100 mg of 4-aminoantibody Pirin (Tokyo Chemical Industry Co., Ltd.) was dissolved in 0.1 M potassium phosphate buffer (pH 7.0). and adjust to 1 L.

[0151] (2) Reagent 2: TOOS solution 500mg of TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine Dissolve sodium (Dojin Chemical Industries, Ltd.) in ion-exchanged water and adjust the volume to 100 ml.

[0152] (3) Reagent 3: Substrate solution (150mM; final concentration 5mM) Dissolve 417 mg of fructosyl valine in ion-exchanged water and adjust the volume to 10 ml.

[0153] B.Measurement method Mix 2.7 ml of Reagent 1, 100 μl of Reagent 2, and 100 μl of Reagent 3. Preheat at ℃ for 5 minutes. Then add 100 μl of enzyme solution, mix well, and measure with a spectrophotometer. The absorbance at 555 nm was measured using a spectrophotometer (U-3010, Hitachi High-Technologies Corporation). The measured value is the change in absorbance per minute at 555 nm from 1 minute to 3 minutes after the start of the experiment. The control solution is prepared by adding 100 μl of ion-exchanged water instead of 100 μl of Reagent 3. The same procedure was used to prepare the solution, except that the solution was prepared in the same manner as above. The number of enzyme molecules is regarded as activity units (U) in the enzyme solution, and is calculated according to the following formula. Activity (U / ml) = {(ΔAs-ΔA0)×3.0×df}÷(39.2×0.5× 0.1) ΔAs: Change in absorbance of the reaction solution per minute ΔA0: Change in absorbance of the control solution per minute 39.2: Quinone imine dyes produced by the reaction Millimolar extinction coefficient (mM -1 ·cm -1 ) 0.5: The number of moles of quinoneimine dye produced by 1 mol of hydrogen peroxide df: Dilution factor

[0154] (Method for measuring surfactant resistance) Add crude amadoriase enzyme solution or purified amadoriase to a concentration of approximately 1.0 U / ml. Sea urchin was diluted with 30 mM MES / 21 mM Tris buffer (pH 6.5) and CTAC (e.g., Tokyo Chemical Industry Co., Ltd.) to a final concentration of 0.01% (w / v) or 0.04%. After adding the above, heat at 30°C for 5 minutes. After heating, add 10 mM Dilute 2-fold with phosphate buffer (pH 7.0) and use the method described above in B. to obtain the sample before surfactant treatment. The enzyme activity of the sample before and after surfactant treatment was measured, and the activity before surfactant treatment was set at 100. The remaining activity (%) was calculated by calculating the percentage of activity after the surfactant treatment. and evaluate surfactant resistance.

[0155] (Buffer evaluation method) In the above-mentioned method for measuring surfactant resistance, 30 mM MES / 21 mM Tris buffer By measuring the residual activity of amadoriase using various buffers instead of buffer solution, The contribution of the agent to the remaining amadoriase activity can be evaluated. Instead of ES / 21 mM Tris buffer (pH 6.5), phosphate buffer (pH 7.0), Use phosphate buffer (pH 6.0), HEPES buffer (pH 7.0), ACES buffer (pH 7.0), etc. Other conditions and procedures can be the same as those in the above-mentioned surfactant resistance measurement method.

[0156] (Stabilizer evaluation method) In the above surfactant resistance measurement method, various stabilizers were further added to the amadori The effect of the stabilizer can be evaluated by measuring the remaining activity of the enzyme. When the target stabilizer is a compound that also has a buffering effect, the buffering effect of the compound To evaluate the amadoriase stabilization effect that is not due to the contribution to the residual amadoriase activity In the present invention, a buffer that does not have an amadoriase stabilizing effect is added in an amount sufficient to provide a buffering capacity to the solution. While the stabilizer is used at a high concentration (e.g., HEPES (pH 7.0) at 500 mM), the stabilizer is used at a low concentration, i.e. It can be used at low concentrations, i.e., not enough to provide a buffering capacity to the solution. A concentration sufficient to provide a pH that does not cause changes in pH due to other reagents added to the solution is It refers to the concentration at which the pH is maintained within a certain range (e.g. pH 5-10, pH 6-8). A concentration that is not sufficient to achieve a certain pH range is one in which the addition of other reagents to the solution would cause pH fluctuations and the pH would fall within a certain range. These concentrations vary depending on the type and amount of other reagents added to the solution. The concentration varies depending on the conditions, but a person skilled in the art can appropriately determine the concentration by a conventional method. The conditions and procedures may be the same as those for the above-mentioned surfactant resistance measurement method.

[0157] (Combination Action) The amadoriase stabilizing effect when the buffer of the present invention and the stabilizer of the present invention are used in combination is evaluated. In order to evaluate the activity of the amadoriase of the present invention, a solution containing the amadoriase and a surfactant was added with a stabilizer and a buffer. It is also possible to add the agent at an appropriate concentration and measure the remaining amadoriase activity. The conditions and procedures for the above-mentioned surfactant resistance measurement method can be the same as those for the above-mentioned surfactant resistance measurement method.

[0158] The present invention will be described in more detail below with reference to examples. is not intended to be limited in any way by these examples.

[0159] [Example 1] (About surfactant-resistant mutations) (1) Preparation of recombinant plasmid pKK223-3-CFP-T7 DNA E. coli JM10 carrying a recombinant plasmid containing the CFP-T7 gene (SEQ ID NO:2) 9(pKK223-3-CFP-T7) strain (see International Publication WO 2007 / 125779). , LB-amp medium [1% (W / V) bactotryptone, 0.5% (W / V) pepto 0.5% (W / V) NaCl, 50 μg / ml Ampicilin] 2.5 ml and cultured at 37°C for 20 hours with shaking to obtain a culture.

[0160] The culture was centrifuged at 7,000 rpm for 5 minutes to collect the bacteria. Next, recombinant DNA was extracted from the cells using QIAGEN tip-100 (Qiagen). The recombinant plasmid pKK223-3-CFP-T7 was extracted and purified to obtain the recombinant plasmid p 2.5 μg of KK223-3-CFP-T7 DNA was obtained.

[0161] (2) Site-specific modification of recombinant plasmid pKK223-3-CFP-T7 DNA Made by Using the resulting recombinant plasmid pKK223-3-CFP-T7 DNA as a template, Synthetic oligonucleotides of SEQ ID NOs: 5 and 6, KOD-Plus- (manufactured by Toyobo Co., Ltd.) were used. The PCR reaction was carried out under the following conditions.

[0162] That is, 5 μl of 10×KOD-Plus-buffer and 2 mM of dNTP were added. 5 μl of the prepared dNTPs mixed solution, 2 μl of 25 mM MgSO4 solution, and template 50 ng of pKK223-3-CFP-T7 DNA and the above synthetic oligonucleotides Add 15 pmol of each and 1 Unit of KOD-Plus-, and dilute the total volume to 5 ml with sterilized water. The reaction solution was then cooled to 0 μl using a thermal cycler (Eppendorf). Incubate at 94°C for 2 minutes, followed by "94°C, 15 seconds" - "50°C, 30 seconds" - The cycle of "68°C, 6 minutes" was repeated 30 times.

[0163] A portion of the reaction mixture was electrophoresed on a 1.0% agarose gel, and a DNA fragment of approximately 6,000 bp was identified. The DNA thus obtained was amplified by restriction enzyme DpnI (N (EW ENGLAND BIOLABS) to cleave the remaining template DNA. After that, E. coli JM109 was transformed and spread on LB-amp agar medium. The LB-amp medium was inoculated with roni and cultured with shaking. The plasmid was then isolated in the same manner as in (1) above. The DNA encoding the amadoriase in the plasmid was isolated. , Multi-capillary DNA analysis system Applied Biosystems 31 Using a 30xl Genetic Analyzer (Life Technologies), As a result, it was found that the asparagine at position 262 of the amino acid sequence shown in SEQ ID NO:1 was replaced by histidine. The recombinant plasmid (pKK223) encoding the modified amadoriase with -3-CFP-T7-262H) was obtained.

[0164] Next, in order to replace valine at position 257 of the amino acid sequence shown in SEQ ID NO: 1 with cysteine, To this end, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template to amplify the sequence The synthetic oligonucleotides Nos. 7 and 8, KOD-Plus- (Toyobo Co., Ltd.), were used. PCR reaction, transformation of E. coli JM109, and growth of colonies were performed under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA was determined. As a result, a modified peptide was obtained in which valine at position 257 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with cysteine. The recombinant plasmid encoding the modified amadoriase (pKK223-3-CFP-T7 -257C).

[0165] Next, in order to replace valine at position 257 of the amino acid sequence shown in SEQ ID NO: 1 with serine, , using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template, The synthetic oligonucleotides 8 and 9, and KOD-Plus- (Toyobo Co., Ltd.) were used. Under similar conditions, PCR reactions, transformation of E. coli JM109, and the plasmids retained by the grown colonies were The base sequence of the DNA encoding amadoriase in the plasmid DNA was determined. As a result, a modified amylase was obtained in which valine at position 257 of the amino acid sequence shown in SEQ ID NO:1 was replaced with serine. The recombinant plasmid encoding the lyase (pKK223-3-CFP-T7-257 S).

[0166] Next, in order to replace valine at position 257 of the amino acid sequence shown in SEQ ID NO: 1 with threonine, To this end, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template to amplify the sequence Synthetic oligonucleotides Nos. 8 and 10 and KOD-Plus- (Toyobo Co., Ltd.) were used. PCR reaction, transformation of E. coli JM109, and growth of colonies were maintained under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA was determined. As a result, valine at position 257 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with threonine. The recombinant plasmid encoding the modified amadoriase (pKK223-3-CFP-T 7-257T).

[0167] Next, the glutamic acid at position 253 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with lysine. To this end, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template to Synthetic oligonucleotides 11 and 12, KOD-Plus- (Toyobo Co., Ltd.) were used. PCR reaction, transformation of E. coli JM109, and growing colonies were performed under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA was determined. As a result, the glutamic acid at position 253 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine. The recombinant plasmid (pKK223-3-CF P-T7-253K) was obtained.

[0168] Next, the glutamic acid at position 253 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with arginine. To do this, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template. , synthetic oligonucleotides of SEQ ID NOs: 12 and 13, KOD-Plus- (manufactured by Toyobo Co., Ltd.) Using the above, PCR reaction, transformation of E. coli JM109, and growth of colonies were performed under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA carried by As a result, it was found that the glutamic acid at position 253 of the amino acid sequence shown in SEQ ID NO: 1 was replaced by arginine. The recombinant plasmid (pKK223- 3-CFP-T7-253R) was obtained.

[0169] Next, in order to replace glutamine at position 337 of the amino acid sequence shown in SEQ ID NO:1 with lysine, To this end, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template to amplify the sequence Synthetic oligonucleotides Nos. 14 and 15, KOD-Plus- (Toyobo Co., Ltd.) were used. PCR reaction, transformation of E. coli JM109, and growing colonies were obtained under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA carrying the gene was determined. As a result, glutamine at position 337 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine. The recombinant plasmid (pKK223-3-CFP- We obtained the following: T7-337K.

[0170] Next, glutamic acid at position 340 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with proline. To do this, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template. Synthetic oligonucleotides of SEQ ID NOs: 16 and 17, KOD-Plus- (manufactured by Toyobo Co., Ltd.) Using the same conditions as above, PCR reaction, transformation of E. coli JM109, and growth of colonies were performed. The base sequence of the DNA encoding amadoriase in the plasmid DNA carried by As a result, glutamic acid at position 340 of the amino acid sequence shown in SEQ ID NO:1 was changed to proline. The recombinant plasmid (pKK223-3- CFP-T7-340P) was obtained.

[0171] Next, glutamic acid at position 133 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with alanine. To do this, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template. Synthetic oligonucleotides of SEQ ID NOs: 18 and 19, KOD-Plus- (manufactured by Toyobo Co., Ltd.) Using the same conditions as above, PCR reaction, transformation of E. coli JM109, and growth of colonies were performed. The base sequence of the DNA encoding amadoriase in the plasmid DNA carried by As a result, glutamic acid at position 133 of the amino acid sequence shown in SEQ ID NO:1 was changed to alanine. The recombinant plasmid (pKK223-3- CFP-T7-133A) was obtained.

[0172] Next, the glutamic acid at position 133 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with methionine. To do this, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template. , synthetic oligonucleotides of SEQ ID NOs: 19 and 20, KOD-Plus- (manufactured by Toyobo Co., Ltd.) Using the above, PCR reaction, transformation of E. coli JM109, and growth of colonies were performed under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA carried by As a result, it was found that the glutamic acid at position 133 of the amino acid sequence shown in SEQ ID NO: 1 was changed to methionine. The recombinant plasmid (pKK223- 3-CFP-T7-133M) was obtained.

[0173] Next, glutamic acid at position 133 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with lysine. To this end, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template to Synthetic oligonucleotides 19 and 21, KOD-Plus- (Toyobo Co., Ltd.) were used. PCR reaction, transformation of E. coli JM109, and growing colonies were performed under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA was determined. As a result, the glutamic acid at position 133 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine. The recombinant plasmid (pKK223-3-CF P-T7-133K) was obtained.

[0174] Next, glutamic acid at position 44 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with proline. To this end, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template to Synthetic oligonucleotides 22 and 23, KOD-Plus- (Toyobo Co., Ltd.) were used. PCR reaction, transformation of E. coli JM109, and growing colonies were performed under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA was determined. As a result, glutamic acid at position 44 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with proline. The recombinant plasmid (pKK223-3-CF P-T7-44P) was obtained.

[0175] Next, in order to replace glycine at position 256 of the amino acid sequence shown in SEQ ID NO: 1 with lysine, Using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template, The synthetic oligonucleotides No. 8 and No. 24, KOD-Plus- (Toyobo Co., Ltd.), PCR reaction, transformation of E. coli JM109, and growth of colonies were performed under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA was determined. As a result, a modification in which glycine at position 256 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine was obtained. The recombinant plasmid (pKK223-3-CFP-T7- 256K).

[0176] Next, glycine at position 256 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with arginine. To this end, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template to Synthetic oligonucleotides in columns 8 and 25, KOD-Plus- (Toyobo Co., Ltd.) were used. PCR reaction, transformation of E. coli JM109, and growing colonies were obtained under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA carrying the gene was determined. As a result, glycine at position 256 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with arginine. The recombinant plasmid (pKK223-3-CFP) encoding the modified amadoriase -T7-256R).

[0177] Next, in order to replace glutamic acid at position 81 of the amino acid sequence shown in SEQ ID NO:1 with lysine, To this end, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template to amplify the sequence Synthetic oligonucleotides Nos. 26 and 27, KOD-Plus- (Toyobo Co., Ltd.) were used. PCR reaction, transformation of E. coli JM109, and growing colonies were obtained under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA carrying the gene was determined. As a result, glutamic acid at position 81 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine. The recombinant plasmid (pKK223-3-CFP- We obtained T7-81K).

[0178] Next, the aspartic acid at position 129 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with lysine. To do this, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template. Synthetic oligonucleotides of SEQ ID NOs: 46 and 47, KOD-Plus- (manufactured by Toyobo Co., Ltd.) Using the same conditions as above, PCR reaction, transformation of E. coli JM109, and growth of colonies were performed. The base sequence of the DNA encoding amadoriase in the plasmid DNA carried by As a result, the aspartic acid at position 129 of the amino acid sequence shown in SEQ ID NO:1 was changed to lysine. The recombinant plasmid (pKK223-3- CFP-T7-129K) was obtained.

[0179] Next, the aspartic acid at position 132 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with lysine. To do this, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template. Synthetic oligonucleotides of SEQ ID NOs: 19 and 48, KOD-Plus- (manufactured by Toyobo Co., Ltd.) Using the same conditions as above, PCR reaction, transformation of E. coli JM109, and growth of colonies were performed. The base sequence of the DNA encoding amadoriase in the plasmid DNA carried by As a result, the aspartic acid at position 132 in the amino acid sequence shown in SEQ ID NO:1 was changed to lysine. The recombinant plasmid (pKK223-3- CFP-T7-132K) was obtained.

[0180] Next, the glutamic acid at position 231 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with lysine. To this end, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template to Synthetic oligonucleotides 49 and 50, KOD-Plus- (Toyobo Co., Ltd.) were used. PCR reaction, transformation of E. coli JM109, and growing colonies were performed under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA was determined. As a result, the glutamic acid at position 231 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine. The recombinant plasmid (pKK223-3-CF P-T7-231K) was obtained.

[0181] Next, the aspartic acid at position 232 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with lysine. To do this, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template. Synthetic oligonucleotides of SEQ ID NOs: 50 and 51, KOD-Plus- (manufactured by Toyobo Co., Ltd.) Using the same conditions as above, PCR reaction, transformation of E. coli JM109, and growth of colonies were performed. The base sequence of the DNA encoding amadoriase in the plasmid DNA carried by As a result, the aspartic acid at position 232 in the amino acid sequence shown in SEQ ID NO:1 was changed to lysine. The recombinant plasmid (pKK223-3- CFP-T7-232K) was obtained.

[0182] Next, the glutamic acid at position 249 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with lysine. To this end, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template to Synthetic oligonucleotides 52 and 53, KOD-Plus- (Toyobo Co., Ltd.) were used. PCR reaction, transformation of E. coli JM109, and growing colonies were performed under the same conditions as above. The base sequence of the DNA encoding amadoriase in the plasmid DNA was determined. As a result, the glutamic acid at position 249 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine. The recombinant plasmid (pKK223-3-CF P-T7-249K) was obtained.

[0183] (3) Production of various modified amadoriases Each of the E. coli JM1 strains carrying the recombinant plasmid obtained by the above procedure The strain 09 was incubated at 30°C in 3 ml of LB-amp medium supplemented with 0.1 mM IPTG. After culturing for 16 hours, each cell was washed with 0.01M phosphate buffer (pH 7.0) and sonicated. The mixture was crushed by shaking and centrifuged at 15,000 rpm for 10 minutes to prepare 1.5 ml of each crude enzyme solution.

[0184] (4) Evaluation of detergent resistance of various modified amadoriases Each crude enzyme solution thus prepared was used as a sample, and the final concentration of CTAC was set to 0.01%. According to the above-mentioned method for measuring the surfactant resistance, the surfactant resistance of each modified amadoriase was measured. The results are shown in Table 1-1. After heating, the sample was diluted 2-fold with BSA solution. The activity was measured again 30 minutes after the incubation, and no change was observed in the activity value. In -1, CFP-T7 was transformed into E. coli JM109 (pKK223-3-CFP-T7) In this example, amadoriase derived from E. coli JM109 (pKK223- 3-CFP-T7) strain-derived amadoriase, CFP-T7, was used as the mutant enzyme. The "amino acid mutations" listed in the table indicate the various mutations that have already been introduced into CFP-T7. is not included. [Table 1-1]

[0185] As shown in Table 1-1, under the conditions of this example, the remaining activity of CFP-T7 was 69.9%. In contrast, 15 mutants obtained by site-directed mutagenesis, i.e., CF In P-T7, asparagine at position 262 was changed to histidine, valine at position 257 was changed to cysteine, and selenium was changed to selenium. Phosphorus, threonine, glutamic acid at position 253, lysine, arginine, glutamic acid at position 337, Glutamic acid at position 340 becomes proline, and glutamic acid at position 44 becomes proline. At position 133, glutamic acid is replaced by alanine, methionine, and lysine, and at position 256, glycine is replaced by phosphorus. lysine, arginine, glutamic acid at position 81 to lysine, asparagine at position 129 acid becomes lysine, aspartic acid at position 132 becomes lysine, and glutamic acid at position 231 becomes lysine. In addition, the 232nd aspartic acid is converted to lysine, or the 249th glutamic acid is converted to lysine. In each mutant amadoriase, the residual activity was increased to more than 72%, which was a significant improvement. In some cases, the improvement was 79% or more, and in more notable cases, the improvement was 89% or more. These mutations improve the detergent resistance of amadoriase. was confirmed.

[0186] In addition, the 253rd and 256th positions of CFP-T7 are basic amino acid residues. The surfactant resistance was improved for both the amino acid substitution with sucrose and the amino acid substitution with arginine. Therefore, the 81st, 129th, 132nd, 133rd, 231st, 232nd, and 249th At positions 331 and 332, the amino acid residues 333 and 334 are also substituted with arginine, a basic amino acid residue, similar to lysine. It is believed that the substitution improves surfactant resistance.

[0187] (5) CcFX from Curvularia clavata SEQ ID NO: 37 is a ketoamine oxidase from Curvularia clavata ( Hereinafter referred to as CcFX) (International Publication No. WO 2004 / 104203) The gene encoding the amino acid sequence of SEQ ID NO:37 (SEQ ID NO:55) was prepared by the conventional method. The cDNA was synthesized by total synthesis of the fragment by PCR (termination codon TA In this case, the 5' end and 3' end of SEQ ID NO:55 each contain an EcoRI site. The cloned gene sequence was used to add a HindIII site and a HindIII site. It was confirmed that the full length amino acid sequence was consistent with the CcFX sequence in Figure 1. In order to express the gene of SEQ ID NO:55 in E. coli, the following procedure was carried out. The gene synthesized above was transformed into a nucleotide sequence containing EcoRI and HindIII sites (Takara Bio). The vector was digested with two types of restriction enzymes and then transformed into pKK-223-3 Vector (GE Healthcare). The recombinant plasmid pKK223 was created by inserting the plasmid into the EcoRI-HindIII site of The -3-CcFX was obtained and transformed into E. coli JM109 strain under the same conditions as above. The JM109(pKK223-3-CcFX) strain was obtained.

[0188] Next, we introduced a mutation into CcFX that improves its surfactant resistance. The 129th, 132nd, 133rd, and 2nd amino acids in the amadoriase (CFP-T7) from the ta genus The positions correspond to positions 31, 232, 249, 253, 256, and 337. CcFX's 129th, 132nd, 133rd, 229th, 230th, 247th, and 251st places Mutations were introduced at positions 111, 112, 113, 114, 115, and 116.

[0189] A recombinant plasmid containing the CcFX gene (SEQ ID NO: 55) was used as the starting plasmid. The E. coli JM109 (pKK223-3-CcFX) strain carrying the vector was subjected to the above-mentioned Various mutants were prepared in the same manner as described in (1) and (2). The primer sequences are as shown in SEQ ID NOs: 56 to 74. The modified amadoriase was produced according to the procedure described in (4). According to the method, except for the detergent treatment conditions, amadoriase was added to 20 mM phosphorus By diluting with potassium phosphate buffer (pH 7.0) and mixing with 0.01% CTAC, The surfactant resistance of various modified amadoriases was evaluated. The results are shown in Table 1-2. Even if the heated sample was diluted 2-fold with BSA solution and the activity was measured again 30 minutes later, It was confirmed that there was no change in activity value. [Table 1-2]

[0190] As shown in Table 1-2, under the conditions of this example, the residual activity of CcFX was 27.4%. In contrast, 12 mutant amadoriases obtained by site-directed mutagenesis showed In the case of the 100%- or 200%-strain, the residual activity was improved to 34% or more, and in the most remarkable case, to 56% or more. In some cases, the improvement was more notable, reaching 64% or more.

[0191] Thus, the mutations that improved the detergent resistance of CFP-T7 were identified as CcFX When the corresponding position of Therefore, the effect of each of these mutations is restricted to amadoriases derived from specific species. By introducing mutations into the corresponding sites, it is possible to identify various amadoriases. Also has the effect of improving surfactant resistance.

[0192] In addition, amadoriase from the genus Coniochaeta and Curvularia clav The ketoamine oxidase from C. ata shares an amino acid sequence identity of about 80%. Amadoriase from the genus Oniochaeta or Curvularia clavat A ketoamine oxidase derived from another species that has an amino acid sequence identity of 80% or more with the ketoamine oxidase derived from a For amadoriase, the surfactant was also synthesized by introducing a mutation into the corresponding position. It is believed that resistance can be improved.

[0193] In addition, substitution of lysine or arginine at positions 251 and 335 of CcFX results in a binding site that is highly conserved in the CcFX domain. The surfactant resistance was improved. , 133, 229, 230, 247, 251, 254, and 335 are basic Substitution of the amino acid residues lysine or arginine improves detergent resistance. The same is true for other types of amadoriases.

[0194] In addition, substitution of alanine or methionine at position 133 of CFP-T7 and substitution of 1 Substitution of alanine at position 33 improved detergent resistance. Position 133 of FP-T7 and position 133 of CcFX are alanine, a hydrophobic amino acid residue; Methionine, valine, isoleucine, leucine, phenylalanine, tryptophan, It is believed that the substitution of lolin improves the surfactant resistance. The same is true for ase.

[0195] Without being bound by any particular theory, it is believed that the mutant amadoriase of the present invention The mechanism of activator resistance is thought to be, for example, as follows. By replacing the acidic amino acids of the enzyme with hydrophobic or basic amino acids, The affinity of the cationic surfactant to the amadoriase decreases, and the amadoriase is degraded by the denaturing effect of the surfactant. In particular, the basic amino acid residues lysine and arginine are considered to be protected from the When a cyclic amine is introduced, the basic amino acid residues repel the cationic surfactant, which further It is believed that the amadoriase is protected from the denaturing effects of detergents.

[0196] These mutations of the present invention are not only effective as single mutations, but also have the potential to be useful as previously known By combining various known mutations and by combining the mutations of the present invention with each other, It is expected that this will contribute to the creation of mutants with practical advantages.

[0197] [Example 2] (Accumulation of mutations that improve detergent resistance) Based on the findings of the surfactant resistance-improving mutations found in Example 1, By accumulating mutations, amadoriase with improved detergent resistance can be obtained. For this purpose, multiple mutants (double mutants, triple mutants, quadruple mutants, quintuple mutants, six We attempted to accumulate mutations that improve detergent resistance by creating multiple mutants (double mutants, 7-fold mutants, and seven-fold mutants).

[0198] SEQ ID NO:3 is a mutant that improves substrate specificity (E98A) and thermostability (F43Y, G Coniochaete 184D, which has three amino acid residues deleted at the carboxyl terminus The amino acid sequence of amadoriase derived from the amadoriase family (hereinafter referred to as "CFP-D") is shown in the sequence It is encoded by gene number 4. The CFP-D gene was inserted into the pKK223-3 vector. The introduced plasmid DNA was used as a template to accumulate mutations that improve detergent resistance. Synthetic oligonucleotides (SEQ ID NOs: 5, 6, 7, 16, 17, 18, 19, 23, 28, 2 9, 30, 31, 32, 33, 46, 47, 50, 51, 52, 54), KOD-Plu PCR was performed using s- (manufactured by Toyobo Co., Ltd.) under the same conditions as in (2) above, and E. coli JM109 Transformation of the strain and growth of colonies encoding amadoriase in the plasmid DNA carried by the strain The base sequence of the DNA encoding the gene was determined.

[0199] This resulted in the production of pKK223, a mutant in which glutamic acid at position 44 was replaced with proline. -3-CFP-D1; glutamic acid at position 44 is replaced by proline and glutamic acid at position 340 is replaced by proline. pKK223-3-CFP-D2;4, a double mutant in which taminic acid was replaced by proline Glutamic acid at position 4 is replaced by proline, and asparagine at position 262 is replaced by histidine and a triple mutant in which glutamic acid at position 340 is replaced by proline, pKK22 3-3-CFP-D3; glutamic acid at position 44 is replaced by proline, and valine at position 257 is replaced by is replaced by cysteine, asparagine at position 262 is replaced by histidine, and pKK223-3-CFP- is a quadruple mutant in which glutamic acid at position 1 is replaced by proline. D4: glutamic acid at position 44 is replaced by proline, and valine at position 257 is replaced by cysteine. The asparagine at position 262 was replaced by histidine, and the glutamic acid at position 340 was replaced by proline. This is a quintuple mutant in which the 232nd aspartic acid is replaced by lysine and the 232nd aspartic acid is replaced by lysine. pKK223-3-CFP-D4 / 232K; glutamic acid at position 44 was replaced by proline The valine at position 257 was replaced with a cysteine, and the asparagine at position 262 was replaced with a histidine. , glutamic acid at position 340 is replaced by proline, and glutamine at position 249 is replaced by pKK223-3-CFP-D4 / 249K, a quintuple mutant in which acids were replaced by lysines; Glutamic acid at position 44 is replaced by proline, and glutamic acid at position 253 is replaced by lysine. The valine at position 257 was replaced by a cysteine, and the asparagine at position 262 was replaced by a histidine. pKK, a quintuple mutant with a substitution and a substitution of glutamic acid at position 340 with proline 223-3-CFP-D5; glutamic acid at position 44 was replaced with proline, and glutamic acid at position 253 was replaced with glutamic acid was replaced by lysine, valine was replaced by cysteine ​​at position 257, and Asparagine is replaced by histidine, and glutamic acid at position 340 is replaced by proline. and pKK223-3, a sextuple mutant in which the aspartic acid at position 129 is replaced by lysine. -CFP-D5 / 129K; glutamic acid at position 44 is replaced by proline, and glutamic acid at position 133 is replaced by proline. glutamic acid at position 253 is replaced by lysine, and glutamic acid at position 257 is replaced by alanine. valine at position 144 is replaced by cysteine, and asparagine at position 262 is replaced by histidine, and pKK223-3, a sextuplicate mutant in which glutamic acid at position 340 is replaced by proline. -CFP-D6; glutamic acid at position 44 is replaced by proline, and glutamic acid at position 133 is replaced by proline. is replaced by alanine, glutamic acid at position 253 is replaced by lysine, and valine at position 257 is replaced by is replaced by cysteine, asparagine at position 262 is replaced by histidine, and A heptagonist with glutamine substituted with lysine and glutamic acid substituted with proline at position 340. The double mutant, pKK223-3-CFP-D7, was obtained.

[0200] Then, E. coli JM109 strain was transformed under the same conditions as above to obtain E. coli JM109(p KK223-3-CFP-D) strain, Escherichia coli JM109 (pKK223-3-CFP-D1 ) strain, E. coli JM109 (pKK223-3-CFP-D2) strain, E. coli JM109 (p KK223-3-CFP-D3) strain, E. coli JM109 (pKK223-3-CFP-D 4) strain, E. coli JM109 (pKK223-3-CFP-D4 / 232K) strain, E. coli J M109 (pKK223-3-CFP-D4 / 249K) strain, E. coli JM109 (pKK 223-3-CFP-D5), E. coli JM109 (pKK223-3-CFP-D5 / 129K), E. coli JM109 (pKK223-3-CFP-D6), E. coli JM1 The 09 (pKK223-3-CFP-D7) strain was obtained.

[0201] The thus obtained E. coli JM109 (p KK223-3-CFP-T7) strain, Escherichia coli JM109 (pKK223-3-CFP-D ) strain, E. coli JM109(pKK223-3-CFP-D1) strain, E. coli JM109(p KK223-3-CFP-D2) strain, Escherichia coli JM109 (pKK223-3-CFP-D 3) strain, E. coli JM109(pKK223-3-CFP-D4) strain, E. coli JM109( pKK223-3-CFP-D4 / 232K) strain, E. coli JM109 (pKK223-3 -CFP-D4 / 249K), E. coli JM109 (pKK223-3-CFP-D5) strain, E. coli JM109 (pKK223-3-CFP-D5 / 129K), E. coli JM1 09 (pKK223-3-CFP-D6) strain, E. coli JM109 (pKK223-3-C The FP-D7) strain was cultured by the above method, and 1.5 ml of crude enzyme solution of each modified amadoriase was obtained. Each of the obtained crude enzyme solutions was used as a sample, and the surfactant-resistant enzymes described in (4) above were used. According to the method for measuring the activity of the surfactant, the surfactant treatment conditions are more severe than those of the conventional method. The mixture was changed to .04% CTAC, and the surfactant resistance of various modified amadoriases was evaluated. The results are shown in Table 2-1. After heating, the samples were diluted 2-fold with BSA solution. The activity was measured again 30 minutes later, and no change was observed in the activity value. [Table 2-1]

[0202] As shown in Table 2-1, under the conditions of this example, the residual activity of CFP-T7 was only 2.2 Under such harsh detergent conditions, the conventional amadoriase It was confirmed that the activity was almost completely lost.

[0203] In contrast, various multiple mutations were prepared by combining the various single mutations confirmed in Example 1. In the mutants, the residual activity was significantly improved. A double nucleotide sequence in which the amino acid at position 340 is replaced by a proline and the glutamic acid at position 340 is replaced by a proline. The residual activity of the mutant was 37.1%, which was improved compared to CFP-T7. , and the survival of the triple mutant in which asparagine at position 262 was replaced by histidine. The activity was 51.4%, which was further improved compared to CFP-T7. The residual activity of the quadruple mutant in which valine at position 257 was replaced by cysteine ​​was 60.7%. This was significantly improved compared to CFP-T7. The remaining activity of the quintuple mutant in which lysine was substituted for lysine was 95.6%. The residual activity of the sextuplet mutant in which glutamic acid at position 133 was replaced by alanine was 99.2%. Furthermore, this mutation was followed by a heptagonist in which glutamine at position 337 was replaced by lysine. The residual activity of the mutant was 100%, which was significantly improved compared to CFP-T7. AC hardly inactivated amadoriase. The residual activity of the quintuple mutant in which the aspartic acid at position 232 was further replaced by lysine was 66.2%, and the glutamic acid at position 249 of the quadruple mutant CFP-D4 was replaced with a lysine. The remaining activity of the quintuple mutant CFP with the substitution of 1.0% was 91.0%. -D5 in a sextuple mutant in which the aspartic acid at position 129 is further replaced by lysine The activity was 98.1%, significantly improved compared to CFP-T7, and Amadori was inhibited by CTAC. There was almost no inactivation of the ase.

[0204] Moreover, each time a mutation accumulates in CFP-D, the resulting surfactants are further enriched in multiple mutants. Resistance is improved stepwise, and the mutation points of the present invention confirmed in Example 1 are appropriately combined. By doing so, it is possible to produce an amadoriase having even better detergent resistance. It became clear.

[0205] Next, the plasmid DNA in which the CcFX gene was inserted into the pKK223-3 vector was used as a template. To improve the resistance to detergents, we accumulated mutants that had improved resistance to detergents, such as CcF instead of CFP-D. The procedure was the same as above, except that the X gene was used. No. 72, 73), KOD-Plus- (manufactured by Toyobo Co., Ltd.), under the same conditions as in (2) above. PCR reaction, transformation of E. coli JM109 strain, and plasmids harbored by grown colonies The base sequence of the DNA encoding amadoriase in the DNA was determined.

[0206] This results in the substitution of an aspartic acid at position 132 with a lysine and a threonine at position 335 with Lysine-substituted mutant pKK223-3-CcFX / 132K / 335K;1 Glutamic acid at position 33 is replaced by alanine, and threonine at position 335 is replaced by lysine. pKK223-3-CcFX / 133A / 335K, a mutant with a 229 glutamic acid residue; pK2 is a mutant in which the 335 threonine is replaced by lysine and the 336 threonine is replaced by lysine. K223-3-CcFX / 229K / 335K; and glutamic acid at position 251 is replaced by lysine. and a mutant pKK223-3 in which threonine at position 335 is replaced by lysine. -CcFX / 251K / 335K was obtained. Then, E. coli JM109 was obtained under the same conditions as above. The strain is transformed, and the transformed strain is cultured by the above-mentioned method to obtain crude fermentation of various modified amadoriases. Each crude enzyme solution was used as a sample, and the enzyme was subjected to the procedure described in (4) above. According to the surfactant resistance measurement method, but for the surfactant treatment conditions, The enzyme was diluted in 20 mM potassium phosphate buffer (pH 7.0) and 0.01% CTAC was added. By mixing, the surfactant resistance of various modified amadoriases was evaluated. The sample after heating was diluted 2-fold with BSA solution and reactivated 30 minutes later. It was confirmed that there was no change in activity value even after the activity measurement. [Table 2-2]

[0207] As shown in Tables 1-2 and 2-2, under the conditions of this example, the residual activity of CcFX was 27. 4%, whereas the respective mutants prepared by combining the various single mutations confirmed in Example 1 In the double mutants, the residual activity was significantly improved. The CcFX double mutant showed improved detergent resistance compared to the FX single mutant. It was confirmed that the effects of mutations in amadoriase enzymes are cumulative, regardless of the type of enzyme. Ta.

[0208] [Example 3-1] (Evaluation of surfactant TTAC) Instead of the surfactant CTAC used in Example 2, tetradecyltrimethylammonium was used. The stability of CFP-D was evaluated using trichloroethylene (TTAC). According to the surfactant resistance measurement method according to Example 1, except for the surfactant treatment conditions amadoriase was diluted with 20 mM potassium phosphate buffer (pH 7.0) to a concentration of 0.0 By mixing 4% TTAC, the detergent resistance of various modified amadoriases was evaluated. The results are shown in Table 3-1. After heating, the samples were diluted 2-fold with BSA solution. The activity was measured again 30 minutes later, and no change was observed in the activity value. [Table 3-1]

[0209] As shown in Table 3-1, under the conditions of this example, the residual activity of CFP-D was 29.2%. I did.

[0210] In contrast, the various multiple mutants prepared in Example 2 all had significant residual activity. Specifically, glutamic acid at position 44 was replaced with proline, and The remaining activity of the double mutant in which glutamic acid at position 1 was replaced by proline was 69.9%. The mutation also resulted in a change from asparagine at position 262 to histidine. The residual activity of the triple mutant in which cytosine was substituted was 85.3%, which was comparable to that of CFP-D. This mutation was further improved by replacing valine at position 257 with cysteine. The residual activity in the double mutant was 91.1%, which was significantly improved compared to CFP-D. Furthermore, this mutation was followed by a quintuple mutant in which glutamic acid at position 253 was replaced by lysine. The activity was 94.9%. Furthermore, this mutation also resulted in the replacement of glutamic acid at position 133 with alanine. The residual activity of the substituted hexamer was 96.4%. Furthermore, this mutation involved the 337th position The residual activity of the heptagonist in which glutamine was replaced by lysine was 100%, whereas that of the CF The results were significantly improved compared to PD, and amadoriase was hardly inactivated by TTAC. Ta.

[0211] Therefore, these amino acid substitutions improve the resistance of amadoriase to TTAC. I found that it is possible to do this.

[0212] [Example 3-2] (Purification of CFP-T7, CFP-D2, and CFP-D7) Using the crude enzymes of CFP-T7, CFP-D2 and CFP-D7 obtained in Examples 1 and 2, The crude enzyme solution was equilibrated with 20 mM potassium phosphate buffer (pH 8.0) for 4 min. 1 of Q Sepharose Fast Flow resin (GE Healthcare) was adsorbed The resin was then washed with 80 ml of the same buffer, followed by 20 ml of 100 mM NaCl. The proteins adsorbed on the resin were eluted with potassium phosphate buffer (pH 8.0) and then purified by HPLC. The fractions exhibiting dryase activity were collected.

[0213] The resulting fractions exhibiting amadoriase activity were purified using Amicon Ultra-15, 30 The mixture was concentrated using 20 mM K NMWL (Millipore). HiLoad 26 / 60 Su equilibrated with potassium phosphate buffer (pH 7.0) The mixture was applied to Perdex 200pg (GE Healthcare) and eluted with the same buffer. Fractions showing amadoriase activity were collected, and purified preparations of wild-type and modified amadoriase were obtained. The purified sample was purified to a single band by SDS-PAGE analysis. It was confirmed that this was the case.

[0214] (Evaluation of various surfactants) The purified enzymes CFP-T7 and CFP-D2 obtained as described above were analyzed using various surfactants. The stability of CFP-D7 was evaluated according to the surfactant resistance measurement method described in Example 1. However, regarding the detergent treatment conditions, amadoriase was treated with 20 mM potassium phosphate. By diluting with ethanol buffer (pH 7.0) and mixing surfactants of various concentrations, various modifications were performed. The surfactant resistance of the modified amadoriase was evaluated. The results are shown in Table 3-2. Even if the sample was diluted 2-fold with BSA solution and the activity was measured again 30 minutes later, the activity value It was confirmed that there was no change in [Table 3-2]

[0215] As shown in Table 3-2, CFP-T7 before the introduction of mutations was stained with OTAB and OTA as surfactants. Except for C, most of the surfactants significantly reduced the activity. The double mutant CFP-D2 was found to be more effective than CFP-T7 with respect to all types of surfactants tested. The seven-fold mutant CFP-D7 also showed excellent detergent resistance. It showed better surfactant resistance than CFP-T7, and in most cases , and had improved detergent resistance compared to the double mutant CFP-D2.

[0216] As shown in Table 3-2, OTAB (C8), DTAB (C12), and TTA are surfactants with different carbon chain lengths. When B (C14) and STAB (C18) were used, the surfactant resistance of both D2 and D7 was improved. Therefore, decyltrimethylammonium bromide with a carbon chain of C10 and hexadecyltride with a carbon chain of C16 are The same can be said for surfactants such as trimethylammonium bromide. The chloride salts corresponding to the bromide salts are OTAC (C8), TTAC (C14), CTAC (C16), and STAC. The same is true for (C18), and the amadoriase of the present invention has a carbon chain of C10, decyltrimethyl For ammonium chloride and dodecyltrimethylammonium chloride with a carbon chain of C12 It is believed to be resistant.

[0217] In addition, from Table 3-2, D2 and D7 have the counter ion (Z) of chloride ion and bromide ion, respectively. Even ions showed resistance to surfactants.

[0218] In addition, from Table 3-2, both D2 and D7 are not only ammonium ion surfactants but also It is also resistant to pyridinium ion surfactants and phosphonium ion surfactants. The surfactant resistance was confirmed to be against various cationic surfactants. It was shown that.

[0219] In summary, the detergent-resistant amadoriase of the present invention is a detergent-resistant amadoriase that is capable of reacting with a counter ion of a detergent. Regardless of the type of surfactant, regardless of the chain length, and regardless of the basic structure of the cationic surfactant, It has been demonstrated that the composition has a broad spectrum of surfactant resistance, regardless of the type of surfactant used.

[0220] The names and structures of the various surfactants used are as follows: [Table 3-3]

[0221] [Example 4] (Evaluation against surfactant SDS) Instead of the surfactant CTAC used in Example 2, sodium dodecyl sulfate (hereinafter, "S The stability of CFP-D was evaluated using the surfactant DS (Figure 1). According to the method for measuring drug resistance, except for the detergent treatment conditions, amadoriase was used for 3 Dilute with 0 mM MES / 21 mM Tris buffer (pH 6.5) and add 0.04% SD The surfactant resistance of various modified amadoriases was evaluated by mixing them with S. The results are shown in Table 4. After heating, the samples were diluted 2-fold with BSA solution and reactivated 30 minutes later. It was confirmed that there was no change in activity value even after the activity measurement. [Table 4]

[0222] As shown in Table 4, under the conditions of this example, the residual activity of CFP-T7 was 2.76%. Ta.

[0223] In contrast, the various multiple mutants prepared in Example 2 all had significant residual activity. Specifically, glutamic acid at position 44 was replaced with proline, and The residual activity of the double mutant in which glutamic acid at position 1 was replaced by proline was 19.2%. The mutation also altered the asparagine at position 262 to a cytochrome P450 variant, which was improved compared to CFP-T7. The residual activity of the triple mutant with stidine substitutions was 11.3%, and the activity of CFP-T7 was 11.3%. In addition, this mutation was followed by a quadruple mutation in which valine at position 257 was replaced with cysteine. The residual activity of the mutant was 17.1%, which was improved compared to CFP-T7. , and the remaining activity in the quintuple mutant in which glutamic acid at position 253 was replaced by lysine The percentage of 133 glutamic acid residues in the 133 position of the 133-glutamic acid residue was 5.07%. The residual activity of the sextuple mutant was 3.62%. Furthermore, this mutation was accompanied by a glutamate at position 337. The residual activity of the heptagonist mutant in which lysine was substituted for cysteine ​​was 3.92%, whereas that of the heptagonist mutant was 3.92%. It's an improvement over the T7.

[0224] Therefore, these amino acid substitutions improve the resistance of amadoriase to SDS. It was found that...

[0225] [Example 5] (Measurement of fructosyl peptide sample in the presence of surfactant) The purified enzymes CFP-T7 and CFP-D7 obtained in Example 3-2 were used to carry out the following The activity of CFP-T7 and CFP-D7 was measured by Amado et al. According to the method for measuring lyase activity, αFVH at a final concentration of 5 mM was used as a substrate, and p Determined using Reagent 1 adjusted to H7.0.

[0226] (11) Preparation of fructosyl peptide samples (Reagent 4) Dissolve 125 mg of αFVH in ion-exchanged water and adjust the volume to 10 ml. This resulted in a 30 mM substrate solution. It was then diluted 714-fold with CTAC solution to give a 4 A 2 μM αFVH / 0%-0.2% CTAC solution was obtained.

[0227] (12) Measurement of fructosyl peptide samples (Reagent 5) 0.21 mM DA-64 (N-(carboxymethylaminocarbonyl)-4,4′-biphenyl (Dimethylamino)diphenylamine sodium, manufactured by Wako Pure Chemical Industries, Ltd.) 20mM potassium phosphate buffer (pH 7.0) (Reagent 6) 6.7U / ml CFP-T7 or CFP-D7 19U / ml peroxidase (Kikkoman) 20mM potassium phosphate buffer (pH 7.0) 135 μl of (Reagent 5) that had been preheated at 37° C. for 5 minutes and the To the 25 μl sample mixture, 50 μl of (Reagent 6) was added to start the reaction. After incubation at ℃ for 5 minutes, the absorbance at 751 nm was measured using the Bio Majest automatic analyzer. y Measured using JCA-BM1650 (JEOL). Ion-exchanged water was used instead of the substrate solution. The same procedure was performed on the (Reagent 4) prepared using the same method, and the measurement was performed at a wavelength of 751 nm. When the absorbance (reagent blank) was used as a control, each sample was measured according to the following formula: The change in absorbance (difference) was calculated. The final concentration of the chromogenic substrate DA-64 was 0.15 mM. When the substrate was used, the final concentration of αFVH was 5 μM, and the length of the cell used for the absorbance measurement ( The tract was 1 cm. Change in absorbance = ΔAes-Ae0 ΔAes: Absorbance 5 minutes after the start of the reaction ΔAe0: Absorbance 5 minutes after the start of the reaction when the control solution was added

[0228] The change in absorbance was calculated when αFVH / 0%-0.2% CTAC was used as a sample. As shown in Table 5. [Table 5]

[0229] As shown in Table 5, under the conditions of this example, when 0% CTAC was mixed, CFP- The change in absorbance of T7 was 0.150, and when mixed with 0.005% CTAC, The change in absorbance of FP-T7 was 0.111. Furthermore, when 0.01% CTAC was mixed, In the experiment, the absorbance change of CFP-T7 was 0.077, and the absorbance change of CFP-T7 was 0.02% with CTAC. Under these conditions, the change in absorbance of CFP-T7 was 0.031, and the change in absorbance of CFP-T7 was 0.04%. When C was mixed, the absorbance change of CFP-T7 decreased to 0.005, and the CTAC concentration It was found that the higher the concentration, the smaller the change in absorbance.

[0230] In contrast, in the case of CFP-D7, when 0.02% CTAC was mixed, CF The change in absorbance of P-D7 was 0.140. The change in absorbance of FP-D7 was 0.069. The more the mixture was mixed, the less the absorbance change, whereas CFP-D7 showed a decrease in the absorbance change after mixing. Furthermore, the absorbance change was constant in the presence of 0.1% or more CTAC. In addition, the absorbance change was small up to 1.3 mM (0.04%), which is the critical micelle concentration of CTAC. The decrease in the amount of α-amyloides is large, but at concentrations above the critical micelle concentration, the effect on amadoriase is small. Therefore, the change in the effect of CFP-D7 was smaller under high CTAC concentration. It was also found that it was stable and that αFVH could be measured.

[0231] [Example 6] (Quantitative analysis of fructosyl peptide samples in the presence of surfactants) Using purified CFP-T7 and CFP-D7 enzymes, 0 The linearity of αFVH measurements was compared in the range of 0.5 to 3 μM. CFP-T7 was 0.01% Or 0.02% CTAC was mixed, and further diluted to 4.2 μM, 8.4 μM, 13 μM, and 17 μM. αFVH at 0.5 μM, 1.0 μM, or 21 μM or 25 μM final concentration The experiments were carried out under conditions using αFVH at 1.5 μM, 2.0 μM, 2.5 μM or 3.0 μM. The change in absorbance was measured in the same manner as in Example 5, and the correlation coefficient was calculated. Mix with 0.02% or 0.2% CTAC and use the same αFVH as above. The change in absorbance was measured in the same manner as in Example 5, and the correlation coefficient was calculated. The results are shown in Table 6. The correlation data are shown in Figures 2, 3, 4, and 5. Figure 2 shows the CF Figure 3 shows the results of measuring αFVH using P-T7, with 0.02% CTAC mixed. Figure 4 shows the results of measuring αFVH using CFP-T7 under the same conditions as in Example 1. Figure 5 shows the results of measuring αFVH using CFP-D7 in the presence of TAC. This is the result of measuring αFVH using CFP-D7 in the presence of 2% CTAC. [Table 6]

[0232] As shown in Table 6, under the conditions of this example, when 0.01% CTAC was mixed, The correlation coefficient of 0.5μM to 3.0μM αFVH with FP-T7 was high at 0.924. However, when CTAC was mixed at 0.02%, the correlation coefficient was low at 0.625. In contrast, when CFP-D7 was used, even in the presence of 0.2% CTAC, the The correlation coefficient for αFVH at ~3.0 μM was 0.965, showing high linearity.

[0233] In addition, the enzyme-based HbA1c measurement kit, Sanku HbA1c (Arkray, Inc.) According to the package insert, the whole blood sample was diluted 416-fold before reacting with amadoriase. For example, if the HbA1c value in the NGSP is 6.5% and the total Hb concentration is 14 The range is 1 to 150 g / l. When blood samples are diluted 416 times, The concentration of excised αFVH is 0.50-0.53 μM. The cutoff for bA1c is 6.5% in the NGSP range. It can be fully utilized in the measurement of HbA1c in the It can be said that the use of TAC in combination can increase the measurement sensitivity.

[0234] [Example 7] (Evaluation of detergent resistance of various amadoriases) An amido group which remains active even in the presence of a surfactant, preferably an ionic surfactant. In order to provide a composition for measuring glycated hemoglobin containing a lyase, We have modified the amadoriase from the genus Ochaete to improve its detergent resistance. In amadoriases other than those from the Oniochaeta genus, ionic It is not known whether HbA1c measurement is possible in the presence of surfactants. Fructosyl peptide oxidase and N from osphaeria nodorum Ketoamine oxidase and iodine from Eocosmospora vasinfecta We attempted to measure αFVH by combining carboxylic acid surfactants.

[0235] 1. Fructosyl peptide oxidoreductase from Phaeosphaeria nodorum Production and purification of enzyme SEQ ID NO: 40 is a fructosyl peptide derived from Phaeosphaeria nodorum The amino acid sequence of PnFX (Biotechnol ogy and Bioengineering, 106, 358-366, 20 The gene encoding the amino acid sequence of SEQ ID NO:40 (SEQ ID NO:44) was cloned by a conventional method. It was obtained by synthesizing cDNA by total synthesis of a gene fragment using PCR. At this time, the 5' and 3' ends of SEQ ID NO:40 contain an NdeI site and a BamHI site, respectively. The full length amino acid sequence predicted from the cloned gene sequence is shown in Figure 1. It was confirmed that the sequence was identical to the PnFX sequence of SEQ ID NO: 44. To express the gene in E. coli, the following procedure was carried out. First, the gene synthesized above was was treated with two restriction enzymes, NdeI and BamHI (Takara Bio), to create pE Inserted into the NdeI-BamHI site of T-22b(+) Vector (Novagen) The recombinant plasmid pET22b-PnFX was obtained by inserting the plasmid into the nuclease, and the plasmid pET22b-PnFX was then transformed into the recombinant plasmid pET22b-PnFX under the same conditions as above. Transform E. coli BL21(DE3) with pET22b -PnFX) strain was obtained.

[0236] The PnFX-producing E. coli BL21(DE3) (pET 22b-PnFX) strain was cultured in LB-am supplemented with IPTG to a final concentration of 0.1 mM. The resulting cultured cells were inoculated into p medium and cultured at 25°C for 16 hours. After washing with sodium buffer (pH 8.0), the cells were suspended in the same buffer and subjected to ultrasonic disruption. The mixture was centrifuged at 20,000×g for 10 minutes to prepare a crude enzyme solution.

[0237] The crude enzyme solution of PnFX prepared was prepared as described in the aforementioned non-patent literature (Biotechnology and d Bioengineering, 106, 358-366, 2010) The crude enzyme solution was fractionated with ammonium sulfate and purified to 10 mM The mixture was dialyzed against potassium phosphate buffer (pH 8.0) and purified by anion exchange chromatography ( In the present embodiment, Q Sepharose Fast Flow was used) and the gel was purified. Filtration chromatography (in this example, HiLoad 26 / 600 Sueprdex The fractions were analyzed by SDS-PAGE and purified using the 200-fold enzyme purification method. The purified PnFX sample was confirmed to be free of contaminating proteins. It was decided.

[0238] 2. Ketoamine oxidase from Neocosmospora vasinfecta Production and refinement SEQ ID NO: 38 is a ketoamine thiol from Neocosmospora vasinfecta The amino acid sequence of NvFX is shown in SEQ ID NO:38. The recombinant plasmid pET22b-NvFX, into which the gene (SEQ ID NO: 45) was inserted, was then transformed with the large intestine. The activity of NvFX has been confirmed by expressing it in bacteria (International Publication No. 2012 / 1 (See Patent Publication No. 8094.) The Escherichia coli BL21(DE3) strain was transformed in the same manner as in Example 1, and the resulting The E. coli BL21(DE3)(pET22b-NvFX) strain was cultured as described above. A crude enzyme solution of NvFX was prepared.

[0239] The crude enzyme solution was equilibrated with 10 mM potassium phosphate buffer (pH 8.0). The sample was then adsorbed onto Sepharose Fast Flow resin (GE Healthcare), and then Wash the resin with 10 mM potassium phosphate buffer (pH 8.0) containing 20 mM NaCl. Then, 10 mM potassium phosphate buffer (pH 8.0) containing 300 mM NaCl was added. The NvFX adsorbed on the resin was eluted and recovered.

[0240] The resulting crude enzyme solution containing NvFX was diluted with 20 mM MES- HiLoad 26 / 600 Superd equilibrated with NaOH buffer (pH 7.0) The mixture was applied to an ex 200 column and NvFX was eluted with the same buffer. Fractions showing acid oxidase activity (amadoriase activity) were collected. Analyzed by S-PAGE and purified to a degree that does not contain other contaminating proteins This was confirmed as a purified preparation of NvFX.

[0241] The various amadoriase purified samples obtained as described above were used as samples, and the same procedures as in Example 5 were carried out. Similarly, we measured αFVH in the presence of CTAC using PnFX and NvFX. The results are shown in Table 7. [Table 7]

[0242] As shown in Table 7, under the conditions of this example, PnFX The change in absorbance was 0.173, and when 0.01% CTAC was mixed, the change in absorbance was The amount of PnFX was 0.115. The change in absorbance for CFP-T7 was 0.031, while that for CFP-T8 was 0.084. When mixed with 0.04% CTAC, the change in absorbance of PnFX was 0.026. The change in absorbance of CFP-T7 was 0.005. The substrate αFVH was measured in the presence of a high concentration of CTAC (0.02% or higher). It can be said that this is possible.

[0243] For NvFX, the absorbance increased when CTAC was mixed up to 0.02%. Due to the influence of turbidity, the absorbance of the blank using ion-exchanged water instead of the substrate was also increased, and αFVH could not be measured accurately.

[0244] [Example 8] (Evaluation of detergent resistance of amadoriase in the presence of various buffers) When using a buffer other than 30 mM MES / 21 mM Tris buffer (pH 6.5), We investigated whether the detergent resistance of amadoriase could be improved by using the above-mentioned purified C FP-T7 was used as the sample, and the final concentration of CTAC was 0.01%. Instead of 21 mM Tris buffer (pH 6.5), various buffers, specifically phosphate and phosphate Phosphate buffer (pH 7.0) containing potassium citrate, citric acid and sodium citrate citrate buffer (pH 6.0) containing MES and its sodium salt; pH 7.0), HEPES buffer containing HEPES and its sodium salt (pH 7.0) In the presence of ACES buffer (pH 7.0) containing ACES and its sodium salt, The surfactant resistance of CFP-T7 was evaluated according to the surfactant resistance measurement method of Example 1. The results are shown in Table 8. After heating, the samples were diluted 2-fold with BSA solution and then 30 minutes later It was confirmed that there was no change in activity value when activity was measured again. [Table 8]

[0245] As shown in Table 8, under the conditions of this example, phosphate buffer, MES buffer, ACES buffer In the presence of the agent, the detergent resistance of CFP-T7 was improved in a buffer concentration-dependent manner. It was revealed that even at 10 mM citrate buffer, the surfactant The HEPES buffer was particularly useful because it did not cause any inactivation of amadoriase. From the above, it was surprising that the phosphate buffer, quencher, and Phosphate buffer, MES buffer, and ACES buffer have the ability to inhibit amadoriase from reacting with detergents. This indicates that it has the effect of improving resistance.

[0246] [Example 9] (Evaluation of surfactant resistance of amadoriase when various stabilizers are added) We investigated whether the addition of various stabilizers improves the detergent resistance of amadoriase. As stabilizers, phosphoric acid, tricarboxylic acids (e.g., citric acid), dicarboxylic acids (e.g., For example, malic acid, maleic acid, citraconic acid, malonic acid, glutaric acid, tartaric acid), monocarboxylic acid, Carboxylic acids (e.g., acetic acid), MES, MOPS, MOPSO, and ammonium sulfate CHES was used as a comparative example. When a stabilizer was added, the pH was prevented from changing. To achieve this, the buffer solution was 500 mM HEPES (pH 7.0), and the solution was purified as described above. The prepared CFP-T7 was used as a sample, and the final concentration of CTAC was set to 0.01%. Various stabilizers were added, and the surface of CFP-T7 was measured according to the surfactant resistance measurement method of Example 1. The surfactant resistance evaluation was performed. The results are shown in Tables 9-1 and 9-2. In addition, 500 mM HEPE Using S (pH 7.0) buffer, the CFP-D2 purified as described above was used as a sample. In addition, various stabilizers were added, and the surfactant resistance measurement method of Example 1 was followed, except that Regarding the surfactant treatment conditions, the final concentration of CTAC was 0.08% and the treatment temperature was 37°C. The surfactant resistance of CFP-D2 was evaluated under severe conditions. The results are shown in Table 9-3. In fact, it was confirmed that the pH actually reached 7.0 when the stabilizer was added. Even if the heated sample was diluted 2-fold with BSA solution and the activity was measured again 30 minutes later, It was confirmed that there was no change in activity value. [Table 9-1] [Table 9-2] [Table 9-3]

[0247] As shown in Tables 9-1 and 9-2, under the conditions of this example, phosphoric acid, quenching agent, and phosphate were used as stabilizers. Acid, malic acid, maleic acid, citraconic acid, malonic acid, glutaric acid, tartaric acid, acetic acid, M When ES, MOPS, MOPSO, and ammonium sulfate were added, the concentration of the stabilizer was dependent on the concentration of the stabilizer. It was found that the surfactant resistance of CFP-T7 was improved by the addition of citric acid. Even when added in a very small amount of 0.2 mM, it was found to improve the surfactant resistance of CFP-T7. As shown in Table 9-3, it was found that phosphoric acid, malic acid, and M Addition of OPS significantly increased the activity of the purified CFP-D2 enzyme compared to the absence of any stabilizer. The surfactant resistance of amadoriase was improved. The stability of amadoriase against surfactants was improved by comparing it with various compounds. It was not known and surprising that the improvement could be achieved by using a substance having the following structure. CHES [ka] had no stabilizing effect, whereas, surprisingly, the closely structurally similar (IV) [ka] [wherein n may be 0, 1, 2 or 3; R 10 are each independently H, OH, -CH2OH or -COOH] MES included in (n=1, R 10 H), MOPS (n=2, R 10 are H), MOPSO( n=2, R 10 Both OH and H) showed amadoriase stabilizing effect. , phosphoric acid, tricarboxylic acid, dicarboxylic acid, monocarboxylic acid, MES, MOPS, MOP The compounds represented by formula (IV) such as SO, ammonium sulfate, have the surfactant activity of amadoriase. In addition, the present invention has been shown to have an effect of improving drug resistance. Regarding amadoriase CFP-T7, the amadoriase CFP into which the mutation of the present invention has been introduced is Improved surfactant resistance was also observed for -D2.

[0248] In addition, taking the results of Tables 8 and 9-1 together, the stabilization of amadoriase by the stabilizer of the present invention is It is clear that this action is different from the amadoriase stabilizing action of the buffer of the present invention. Specifically, Table 8 shows that when MES is used as the buffer of the present invention, CFP-T7α at a concentration of 50 mM The residual activity of Madoriase was 14.3% at a concentration of 100 mM, 17.6% at a concentration of 1 At 50 mM, the residual activity was confirmed to be 62.9%. HEPES (pH 7.0) was used as a simple pH buffer without lyase stabilizing effect, while MES When used as the stabilizer of the present invention, the remaining activity of CFP-T7 amadoriase was reduced at a MES concentration of 10 mM. The residual activity was 19.5% at a MES concentration of 20 mM, and 54.1% at a MES concentration of 20 mM. Even at low concentrations such as 20 mM, where the buffer capacity is not fully exerted, MES has an amadoriase stabilizing effect. The residual activity of amadoriase confirmed was surprisingly high when used as the buffer of the present invention. The residual activity exceeded that of MES at a concentration of 100 mM (Table 8, 17.6%). The amadoriase stabilizing effect of the above stabilizer is comparable to that of the buffer of the present invention. The same is true for phosphoric acid and citric acid. In addition, the dicarboxylic acids, MOPS and MOPSO, which showed stabilizing effects, It can also be used as a buffering agent, so it can be considered the same.

[0249] (Evaluation of surfactant resistance of PnFX when various stabilizers are added) Examples of amadoriase other than amadoriase derived from the genus Coniochaeta include We investigated whether the above stabilizers have the effect of improving surfactant resistance for PnFX. The same as above is used, and in order to prevent pH changes when a stabilizer is added, The buffer used was 300 mM HEPES (pH 7.0), and the purified Pn FX was used as the sample, and the final concentration of CTAC was set to 0.04%, and the same procedure was repeated for PnFX. Surfactant resistance evaluation was performed. In fact, when the stabilizer was added, the pH was actually 7.0. The results are shown in Table 10. After heating, the sample was diluted 2-fold with BSA solution. Even when the activity was measured again 30 minutes after dilution, it was confirmed that there was no change in the activity value. [Table 10]

[0250] As shown in Table 10, under the conditions of this example, the same effect was observed for PnFX as for CFP-T7. When phosphoric acid, citric acid, malic acid, acetic acid, MES, and ammonium sulfate were added, the interface The effect of improving the resistance to surfactants was shown. Monocarboxylic acids, MES, and ammonium sulfate have a wide variety of surfactants for amadoriase. It can be said that it is useful as a stabilizer that improves drug resistance. The amino acid sequence identity is 75%, so it has 75% amino acid sequence identity with CFP-T7. It can be said that an amadoriase having this function has the above-mentioned effects. [Sequence List Free Text]

[0251] SEQ ID NO: 1. Amino acid sequence of CFP-T7 SEQ ID NO: 2. Gene sequence of CFP-T7 SEQ ID NO: 3. Amino acid sequence of CFP-D SEQ ID NO: 4. Gene sequence of CFP-D SEQ ID NO: 5. N262H introduction primer Fw SEQ ID NO: 6. N262X introduction primer Rv SEQ ID NO: 7. V257C introduction primer Fw SEQ ID NO: 8. V257X introduction primer Rv SEQ ID NO: 9. V257S introduction primer Fw SEQ ID NO: 10. V257T introduction primer Fw SEQ ID NO: 11. E253K introduction primer Fw SEQ ID NO: 12. E253X introduction primer Rv SEQ ID NO: 13. E253R introduction primer Fw SEQ ID NO: 14. Q337K introduction primer Fw SEQ ID NO: 15. Q337X introduction primer Rv SEQ ID NO: 16. E340P introduction primer Fw SEQ ID NO: 17. E340X introduction primer Rv SEQ ID NO: 18. E133A introduction primer Fw SEQ ID NO: 19. E133X introduction primer Rv SEQ ID NO: 20. E133M introduction primer Fw SEQ ID NO: 21. E133K introduction primer Fw SEQ ID NO: 22. E44P introduction primer Fw SEQ ID NO: 23. E44X introduction primer Rv SEQ ID NO: 24. G256K introduction primer Fw SEQ ID NO: 25. G256R introduction primer Fw SEQ ID NO: 26. E81K introduction primer Fw SEQ ID NO: 27. E81X introduction primer Rv SEQ ID NO: 28. F43Y / E44P introduction primer Fw SEQ ID NO: 29. V257X / N262H introduction primer Rv SEQ ID NO: 30. E253K / V257C introduction primer Fw SEQ ID NO: 31. E253X / V257C / N262H introduction primer Rv SEQ ID NO: 32. Q337K / E340P introduction primer Fw SEQ ID NO: 33. Q337X / E340P introduction primer Rv SEQ ID NO: 34. Amadoriase from Eupenicillium terrenum SEQ ID NO: 35. Ketoamine oxidase from Pyrenochaeta sp. SEQ ID NO: 36. Ketoamine oxidase from Arthrinium sp. SEQ ID NO: 37. Ketoamine oxidase from Curvularia clavata SEQ ID NO: 38. Ketoamine from Neocosmospora vasinfecta Oxidase SEQ ID NO: 39. Fructosyl from Cryptococcus neoformans Amino acid oxidase SEQ ID NO: 40. Fructosyl peptide from Phaeosphaeria nodorum Thiol oxidase SEQ ID NO: 41. Fructosyl amino acid sequence from Aspergillus nidulans Acid oxidase SEQ ID NO: 42. Fructosyl amino acid oxidase from Ulocladium sp. Z SEQ ID NO: 43. Fructosyl acylate from Penicillium crysogenum Amino acid oxidase SEQ ID NO: 44. Fructosyl peptide from Phaeosphaeria nodorum The gene for thiol oxidase SEQ ID NO: 45. Ketoamine from Neocosmospora vasinfecta Oxidase genes SEQ ID NO: 46. D129K introduction primer Fw SEQ ID NO: 47. D129K introduction primer Rv SEQ ID NO: 48. D132K introduction primer Fw SEQ ID NO: 49. E231K introduction primer Fw SEQ ID NO: 50. E231X introduction primer Rv SEQ ID NO: 51. D232K introduction primer Fw SEQ ID NO: 52. E249K introduction primer Fw SEQ ID NO: 53. E249K introduction primer Rv SEQ ID NO: 54. E249K / V257C introduction primer Rv SEQ ID NO: 55. Ketoamine oxidase from Curvularia clavata Genes SEQ ID NO: 56. Primer Fw for introduction of D129K in CcFX SEQ ID NO: 57. Primer Rv for introducing D129K into CcFX SEQ ID NO: 58. CcFX D132K introduction primer Fw SEQ ID NO: 59. Primer Fw for introducing E133K into CcFX SEQ ID NO: 60. E133A introduction primer Fw of CcFX SEQ ID NO: 61. E133X introduction primer Rv of CcFX SEQ ID NO: 62. Primer Fw for introducing E229K into CcFX SEQ ID NO: 63. D230K introduction primer Fw of CcFX SEQ ID NO: 64. D230X introduction primer Rv of CcFX SEQ ID NO: 65. Primer Fw for introducing E247K into CcFX SEQ ID NO: 66. Primer Rv for introducing E247K into CcFX SEQ ID NO: 67. Primer Fw for introducing E251K into CcFX SEQ ID NO: 68. E251X introduction primer Rv of CcFX SEQ ID NO: 69. CcFX E251R introduction primer Fw SEQ ID NO: 70. Primer Fw for introducing N254K into CcFX SEQ ID NO: 71. N254K introduction primer Rv of CcFX SEQ ID NO: 72. CcFX T335K introduction primer Fw SEQ ID NO: 73. T335X introduction primer Rv of CcFX SEQ ID NO: 74. CcFX T335R introduction primer Fw

[0252] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. This will be incorporated into the.

Claims

1. The residual activity (%) 5 minutes after addition of the surfactant was It was found that the amadoriase had an improved amino acid sequence compared to the amadoriase having the amino acid sequence shown in hand, (i) a deletion of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1, 3, or 37; , an amino acid sequence having insertions, additions, and / or substitutions; and / or (ii) at least 70% identity with the amino acid sequence set forth in SEQ ID NO: 1, 3, or 37; having an amino acid sequence The amadoriase.

2. The amadoriase according to claim 1, wherein the surfactant is an ionic surfactant.

3. The following (i) to (xiv) in the amino acid sequence shown in SEQ ID NO: 1 or 3: (i) an asparagine at position 262, (ii) valine at position 257, (iii) glutamic acid at position 249 (iv) glutamic acid at position 253, (v) glutamine at position 337, (vi) glutamic acid at position 340, (vii) aspartic acid at position 232, (viii) aspartic acid at position 129, (ix) aspartic acid at position 132, (x) glutamic acid at position 133, (xi) glutamic acid at position 44, (xii) glycine at position 256, (xiii) glutamic acid at position 231, and (xiv) glutamic acid at position 81, one or more amino acid residues at positions corresponding to amino acids selected from the group consisting of The amadoriase according to claim 1 or 2, having the following substitution:

4. The amino acid sequence shown in SEQ ID NO: 1 or 3 is Substitutions (i) to (xiv) below: (i) the asparagine at position 262 is substituted with histidine; (ii) valine at position 257 is replaced with cysteine, serine, or threonine; (iii) glutamic acid at position 249 is replaced with lysine or arginine; (iv) glutamic acid at position 253 is replaced with lysine or arginine; (v) glutamine at position 337 is replaced with lysine or arginine; (vi) glutamic acid at position 340 is replaced with proline; (vii) aspartic acid at position 232 is substituted with lysine or arginine; (viii) aspartic acid at position 129 is substituted with lysine or arginine; (ix) aspartic acid at position 132 is substituted with lysine or arginine; (x) glutamic acid at position 133 is replaced by alanine, methionine, lysine, or arginine There are; (xi) glutamic acid at position 44 is replaced by proline; (xii) glycine at position 256 is substituted with lysine or arginine; (xiii) glutamic acid at position 231 is substituted with lysine or arginine; and (xiv) glutamic acid at position 81 is substituted with lysine or arginine; The amadoriase according to any one of claims 1 to 3, having any one or more of the following:

5. In the amino acid sequence shown in SEQ ID NO: 1 or 3, the following (i) to (ix): (i) Substitution of the amino acid at position 44 corresponding to glutamic acid with proline and substitution of the amino acid at the position corresponding to glutamic acid at position 1 with proline; (ii) substitution of the amino acid at position 44 corresponding to glutamic acid with proline, A substitution of the amino acid at the position corresponding to asparagine with histidine and a substitution of glutamic acid at position 340 substitution of the amino acid at the position corresponding to carboxylic acid with proline; (iii) substitution of the amino acid at the position corresponding to glutamic acid at position 44 with proline; substitution of the amino acid at position 262 corresponding to valine with cysteine; and the amino acid at position 340 corresponding to glutamic acid was replaced with histidine. Substitution of the amino acid at position 14 with proline; (iv) substitution of the amino acid at position 44 corresponding to glutamic acid with proline, Substitution of the amino acid at the position corresponding to valine with cysteine, corresponding to asparagine at position 262 substitution of the amino acid at position 340 corresponding to glutamic acid with histidine; and the substitution of the amino acid at position 232 corresponding to aspartic acid at position 233 with proline. Substitution with lysine; (v) substitution of the amino acid at position 44 corresponding to glutamic acid with proline, Substitution of the amino acid at the position corresponding to valine with cysteine, corresponding to asparagine at position 262 substitution of the amino acid at position 340 corresponding to glutamic acid with histidine; The amino acid at position 249 was replaced by proline and the amino acid at position 250 was replaced by glutamic acid at position 249. Substitution for gin; (vi) substitution of the amino acid at position 44 corresponding to glutamic acid with proline, Substitution of the amino acid at the position corresponding to glutamic acid with lysine, corresponding to valine at position 257 substitution of the amino acid at position 262 with cysteine; and the substitution of the amino acid protease at the position corresponding to glutamic acid at position 340. Substitution for phosphorus; (vii) substitution of the amino acid at position 44 corresponding to glutamic acid with proline; substitution of the amino acid at position 257 corresponding to glutamic acid with lysine; substitution of the amino acid at position 262 with cysteine; substitution of histidine for the amino acid at position 340 corresponding to glutamic acid; and a substitution of the amino acid at the position corresponding to aspartic acid at position 129 with lysine. ； (viii) substitution of the amino acid at the position corresponding to glutamic acid at position 44 with proline; substitution of the amino acid at the position corresponding to glutamic acid in position 253 with alanine; substitution of the amino acid at position 257 corresponding to valine with lysine; and a substitution of histidine at the amino acid position corresponding to asparagine 262. and substitution of the amino acid at position 340 corresponding to glutamic acid at position 340 with proline. ;and (ix) substitution of the amino acid at position 44 corresponding to glutamic acid with proline; Substitution of the amino acid at the position corresponding to glutamic acid with alanine, glutamic acid at position 253 Substitution of the amino acid at the corresponding position with lysine, and the amino acid at the position corresponding to valine at position 257 to cysteine, and histidine at the amino acid position corresponding to asparagine at position 262. substitution of the amino acid at the position corresponding to glutamine at position 337 to lysine; Substitution of the amino acid at the position corresponding to glutamic acid at position 0 with proline The method according to any one of claims 1 to 4, further comprising substituting an amino acid residue selected from the group consisting of: The amadoriase described herein.

6. The amino acid sequence shown in SEQ ID NO:37 is Substitutions (i) through (ix) below: (i) glutamic acid at position 247 is replaced with lysine or arginine; (ii) glutamic acid at position 251 is replaced with lysine or arginine; (iii) Threonine at position 335 is replaced with lysine or arginine; (iv) aspartic acid at position 230 is replaced with lysine or arginine; (v) aspartic acid at position 129 is replaced with lysine or arginine; (vi) aspartic acid at position 132 is substituted with lysine or arginine; (vii) glutamic acid at position 133 is replaced with alanine, methionine, lysine, or arginine It is being (viii) asparagine at position 254 is substituted with lysine or arginine; and (ix) glutamic acid at position 229 is substituted with lysine or arginine; The amadoriase according to any one of claims 1 to 3, having any one or more of the following:

7. In the amino acid sequence shown in SEQ ID NO: 37, the following (i) to (iv): (i) a substitution of the amino acid at the position corresponding to glutamic acid at position 251 with lysine and substitution of lysine for the amino acid at the position corresponding to threonine at position; (ii) substitution of the amino acid at the position corresponding to aspartic acid at position 132 with lysine and Substitution of the amino acid at the position corresponding to threonine at position 5 with lysine; (iii) substitution of the amino acid at position 133 corresponding to glutamic acid with alanine and Substitution of the amino acid at the position corresponding to threonine at position 35 with lysine; and (iv) substitution of the amino acid at position 229 corresponding to glutamic acid with lysine and substitution of the amino acid at the position corresponding to the threonine at position 14 with lysine; Any one of claims 1 to 3 and 6, having a substitution of an amino acid residue selected from the group consisting of:

1. An amadoriase according to claim 1.

8. An amadoriase gene encoding the amino acid sequence according to any one of claims 1 to 7. 。

9. A recombinant vector comprising the amadoriase gene according to claim 8.

10. A host cell comprising the recombinant vector of claim 9.

11. The following steps: (i) culturing the host cell of claim 10; (ii) expressing the amadoriase gene contained in the host cell; and (iii) producing amadoriase, comprising isolating the amadoriase from the culture. method.

12. A method for measuring glycated hemoglobin comprising the amadoriase according to any one of claims 1 to 7. Composition for.

13. A composition for measuring glycated hemoglobin, comprising one or more surfactants and amadoriase.

14. Amadoriase is (i) The residual activity (%) 5 minutes after the addition of the surfactant was compared to that without the surfactant. , 15% or more remaining, and / or (ii) In the presence of a surfactant at a final concentration of 0.04%, the chromogenic substrate N-(carboxymethyl) (dimethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine sodium After adding glycated amine (DA-64) and reacting for 5 minutes, the absorbance at 751 nm and the Five minutes after addition of the control solution, which used ion-exchanged water instead of the acid solution or glycated peptide solution The difference between the absorbance at 751 nm of The composition of claim 13.

15. Amadoriase having at least 7 amino acid sequences as set forth in SEQ ID NO: 1, 3, 37 or 40 15. An amadoriase having an amino acid sequence with 0.0% identity to the amadoriase of claim 13 or 14. The composition described in

16. 16. The surfactant according to claim 13, wherein the surfactant has a critical micelle concentration of 70 mM or less. The composition according to any one of claims 1 to 5.

17. The surfactant is a quaternary ammonium salt represented by the following general formula (I): 【Chemistry 1】 [In the formula, R １ ~R ４ may be the same or different, and may be substituted or unsubstituted C １ ~C ２ ０ represents alkyl, alkenyl, aryl or benzyl; Z － represents a monovalent anion 。］ A pyridinium salt represented by the following general formula (II): 【Chemistry 2】 [In the formula, R ５ is a substituted or unsubstituted C １ ~C ２０ represents alkyl; ａ is the same or may be different, and may be a hydrogen atom or a substituted or unsubstituted C １ ~C ２０ Alkyl, ar represents an aryl, aryl or benzyl group, n represents an integer of 1 to 5, Z － is a monovalent anion ] A phosphonium salt represented by the following general formula (III): 【Chemistry 3】 [In the formula, R ６ ~R ９ may be the same or different, and may be substituted or unsubstituted C １ ~C ２ ０ represents alkyl, alkenyl, aryl or benzyl; Z － represents a monovalent anion 。］ and one or more ionic surfactants selected from the group consisting of sodium dodecyl sulfate. The composition according to any one of claims 13 to 16.

18. The surfactant is octyltrimethylammonium chloride, octyltrimethylammonium Dioctyldimethylammonium bromide, dioctyldimethylammonium chloride, dioctyldimethylammonium ammonium bromide, decyltrimethylammonium chloride, decyltrimethylammonium Dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium Bromide, Tetradecyltrimethylammonium chloride, Tetradecyltrimethyla ammonium bromide, hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium Methyl ammonium bromide, octadecyl trimethyl ammonium chloride, octadecyl Cyltrimethylammonium bromide, eicosyltrimethylammonium chloride and Eicosyltrimethylammonium bromide, benzyldodecyldimethylammonium chloride Benzyldodecyldimethylammonium bromide, benzyltetradecyldimethylammonium bromide Benzyl ammonium chloride, benzyl tetradecyl dimethyl ammonium bromide, benzyl Benzylcetyldimethylammonium chloride, and benzylcetyldimethylammonium bromide Romid, 1-Decylpyridinium chloride, 1-Decylpyridinium bromide, 1-Dodecylpyridinium pyridinium chloride, 1-dodecylpyridinium bromide, 1-tetradecylpyridinium 1-tetradecylpyridinium bromide, 1-hexadecylpyridinium chloride Bromide, 1-hexadecylpyridinium bromide, N-cetyl-2-methylpyridinium Chloride, N-cetyl-2-methylpyridinium bromide, N-cetyl-3-methylpyridinium pyridinium chloride, N-cetyl-3-methylpyridinium bromide, N-cetyl-4-methylpyridinium Cetylpyridinium chloride, N-cetyl-4-methylpyridinium bromide, 1-octylpyridinium chloride, Decylpyridinium chloride, 1-octadecylpyridinium bromide, 1-eicosyl Pyridinium chloride and 1-eicosylpyridinium bromide, Tetraethylphosphonium chloride, Tetraethylphosphonium bromide, Tributyl Methylphosphonium chloride, Tributylmethylphosphonium bromide, Tributylmethyl butylphosphonium iodide, tetrabutylphosphonium chloride, tetrabutylphosphonium bromide, tetra-n-octylphosphonium chloride, tetra-n-octylphosphonium nium bromide, tributyldodecylphosphonium chloride, tributyldodecylphosphonium nium bromide, tributylhexadecylphosphonium chloride, tributylhexadecyl methyltriphenylphosphonium bromide, methyltriphenylphosphonium chloride, methyltriphenyl methyltriphenylphosphonium bromide, methyltriphenylphosphonium iodide, tetraphenylphosphine phosphonium chloride, and tetraphenylphosphonium bromide, 18. The composition of claim 17, wherein the surfactant is one or more ionic surfactants selected from the group consisting of: thing.

19. The surfactant contained in the solution is at a final concentration of 0.01% (w / v) or more at the time of measurement. The composition according to any one of claims 13 to 18.

20. Borate buffer, Tris-HCl buffer, phosphate buffer, citrate buffer, fumarate buffer, Glutaric acid buffer, citraconic acid buffer, mesaconic acid buffer, malonic acid buffer, tartaric acid buffer Buffers, succinic acid buffer, adipic acid buffer, ACES (N-(2-acetamido)-2-aminoethyl ethanesulfonic acid) buffer, BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) N,N-bis(2-hydroxyethyl)glycine) buffer, Bis -Tris (bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane) buffer , EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid) buffer, HE PPSO (N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid) buffer agent, MES (2-(N-morpholino)ethanesulfonic acid) buffer, MOPS (3-(N-morpholino) propanesulfonic acid) buffer, MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid) Buffers: piperazine-N,N'-bis(2-ethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) buffers, PO PSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid)) buffer, TAPS (N -Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer, TAPSO ( 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid) buffer agent, TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffer, Tricine (N-tris(hydroxymethyl)methylglycine) buffers and combinations thereof The glycosylated hemoglobin according to claim 13, further comprising one or more buffers selected from the group consisting of: Composition for measuring .

21. Phosphate buffer with a final concentration of 100 mM or more in the measurement solution, 0 mM or more of citrate buffer, and MES (2-(N -morpholino)ethanesulfonic acid) buffer, final concentration in the measurement solution is 100 mM or more OPS (3-(N-morpholino)propanesulfonic acid) buffer, final concentration in the measurement solution is 100mM MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid) buffer, which is greater than or equal to M, ACES (N-(2-acetamido)-2-aminoethoxyethyl ester) was used as the final concentration in the measurement solution at 200 mM or more.

21. The method of claim 20, further comprising one or more buffers selected from the group consisting of: The composition described above.

22. Phosphoric acid, tricarboxylic acid, dicarboxylic acid, monocarboxylic acid, compound of formula (IV), 【Chemistry 4】 [wherein n may be 0, 1, 2 or 3; R 10 are each independently H, OH, or -CH 2 OH or -COOH] and one or more stabilizers selected from the group consisting of ammonium sulfate and combinations thereof. The composition for measuring glycated hemoglobin according to claim 13,

23. The tricarboxylic acid is citric acid, or the dicarboxylic acid is fumaric acid, glutaric acid, Citraconic acid, mesaconic acid, malonic acid, tartaric acid, succinic acid, adipic acid, maleic acid, or the monocarboxylic acid is selected from the group consisting of acetic acid, acetic acid, and combinations thereof. or the compound of formula (IV) is MES, MOPS, MOPSO and combinations thereof.

23. The composition of claim 22, wherein the composition is selected from the group consisting of:

24. The stabilizer is a phosphoric acid having a final concentration of 2 mM or more in the measurement solution, Citric acid with a final concentration of 0.2 mM or more, malic acid with a final concentration of 2 mM or more in the measurement solution, Maleic acid with a final concentration of 2 mM or more in the measurement solution, citraconic acid, malonic acid, and Glutaric acid with a final concentration of 2 mM or more in the measurement solution, and tartaric acid with a final concentration of 2 mM or more in the measurement solution acetic acid having a final concentration of 10 mM or more in the measurement solution; MES (2-(N-morpholino)ethanesulfonic acid), the final concentration in the measurement solution is 10 mM or more MOPS (3-(N-morpholino)propanesulfonic acid), the final concentration in the measurement solution is 10 MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid) at ≥ 1 mM, measurement solution ammonium sulfate having a final concentration of 2 mM or more at 24. The composition of claim 22 or 23, wherein one or more stabilizers are selected.

25. A buffer according to claim 20 or 21 and a stabilizer according to claim 22, 23 or 24. A composition for measuring glycated hemoglobin comprising an agent.

26. amadoriase having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:37 or SEQ ID NO:40 or the amadoriase according to any one of claims 1 to 7. The composition according to any one of claims 20 to 25.

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