JP6068916B2 - Reagent composition for measuring glycated hemoglobin and method for measuring glycated hemoglobin - Google Patents

Description

  The present invention relates to a reagent composition effective for measurement of glycated hemoglobin (HbA1c), more specifically, a reagent composition for measuring glycated hemoglobin which is hardly affected by coexisting ε-fructosyl lysine, and glycated hemoglobin measurement using the same Regarding the method.

  Glycated proteins are produced by non-enzymatic covalent bond formation between the aldehyde groups of aldoses (monosaccharides and their derivatives that potentially have aldehyde groups) such as glucose, and Amadori transfer It is. Examples of the amino group of the protein include an α-amino group at the amino terminal and an ε-amino group on the side chain of a lysine residue in the protein. Known glycated proteins generated in vivo include glycated hemoglobin in which hemoglobin in blood is glycated, glycated albumin in which albumin is glycated, and the like.

  Among these glycated proteins generated in vivo, glycated hemoglobin (HbA1c) has attracted attention as an important blood glucose control marker for diagnosis and symptom management of diabetic patients in the clinical diagnosis field of diabetes. Hemoglobin is a protein composed of a total of 4 subunits, 2 molecules of α subunit (hereinafter referred to as α chain) and 2 molecules of β subunit (hereinafter referred to as β chain), and HbA1c has an amino terminus of β chain. It is defined as glycated hemoglobin. The HbA1c concentration in the blood reflects the average blood glucose level over a certain period in the past, and the measured value is an important index in the diagnosis and management of diabetes symptoms.

As a method for measuring HbA1c quickly and simply, an enzymatic method using amadoriase, that is, α-fructosylvalylhistidine (hereinafter referred to as αFVH) obtained by decomposing HbA1c with a protease and releasing it from its β-chain amino terminus. Or α-fructosyl valine (hereinafter referred to as αFV) has been proposed (see, for example, Patent Documents 1 to 7).
Actually, in the method of cutting out αFV from HbA1c, it is considered that the influence of impurities and the like is large. Therefore, at present, a method of measuring HbA1c by liberating αFVH from the β chain of HbA1c using a specific protease and quantifying it is the mainstream.

Amadoriase catalyzes a reaction that oxidizes iminodiacetic acid or a derivative thereof (also referred to as “Amadori compound”) in the presence of oxygen to produce glyoxylic acid or α-ketoaldehyde, amino acid or peptide, and hydrogen peroxide. To do.
Amadoriase has been found from bacteria, yeasts and fungi, but amadoriase having enzyme activity against αFVH and / or αFV, which is particularly useful for the measurement of HbA1c, includes, for example, the genus Coniochaeta, Eupenicillium ( Eupenicillium genus, Pyrenochaeta genus, Arthrinium genus, Curvularia genus, Neocosmospora genus, Cryptococcus genus, Pheosphaeria genus, Aspergillus genus Emericella, Ulocladium, Penicillium, Fusarium, Achaetomiella, Achaetomium, Thielavia, Chaetomium, Chaetomium Gelasinospora), Microascus Leptosphaeria genus, Ophiobolus genus, Pleospora genus, Coniochaetidium genus, Pichia genus, Corynebacterium genus, Agrobacterium genus, Arthrobacter Amadoriase derived from the genus (Arthrobacter) has been reported (see, for example, Patent Documents 1 and 6 to 15 and Non-Patent Documents 1 to 9). In the above reported examples, amadoriase may be described in terms of ketoamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, fructosylvalylhistidine oxidase, fructosylamine oxidase, etc. depending on the literature. is there.

  In the measurement of HbA1c by an enzymatic method, strict substrate specificity is required as a property of amadoriase. For example, as described above, when the measurement of HbA1c is performed by quantifying αFVH released by a specific protease, the HbA1c is present in a free state in the sample and / or HbA1c using a protease or the like is used. It is desirable to use an amadoriase that is difficult to act on a glycated amino acid or glycated peptide other than αFVH that is released in the treatment step. In particular, it is known that the ε-position amino group of the lysine residue side chain contained in the hemoglobin molecule undergoes glycation, and the ε-position amino group derived from this glycated lysine residue is glycated. It has been suggested that ε-fructosyl lysine (hereinafter referred to as εFK) is released by protease treatment or the like (see Non-Patent Document 10). For this reason, an amadoriase with high substrate specificity that is unlikely to act on εFK, which can be a causative substance of measurement errors, is strongly desired.

  Note that a peptide fragment released from the α-chain amino terminus of HbA1c by a protease that liberates αFVH from the β-chain amino terminus of HbA1c has not been identified so far, and valine at the α-chain amino terminus of hemoglobin is β It has been shown that glycation is extremely difficult compared to valine at the chain amino terminal (see Non-Patent Document 10). Therefore, there is almost no possibility that a glycated amino acid / glycated peptide derived from the α-chain amino terminus can cause a measurement error.

  As a general technique, in order to modify the substrate specificity of an enzyme, mutation is added to the DNA encoding the enzyme, substitution is introduced into the amino acid of the enzyme, and an enzyme having the desired substrate specificity is selected. The method is known. In addition, when an example in which substrate specificity is increased by amino acid substitution in an enzyme having high homology is known, it is possible to predict improvement in substrate specificity based on the information.

  In fact, the ketoamine oxidase derived from Curvularia clavata YH923 and the ketoamine oxidase derived from Neocosmospora vasinfecta 474 have substrate specificity for αFVH by substituting several amino acids. An improved modified ketoamine oxidase has been shown (see Patent Document 1). For example, in the ketoamine oxidase derived from the carbularia clavater YH923, ε-fructosyl- (α-benzyl) is obtained by substituting isoleucine at position 58 with valine, arginine at position 62 with histidine and phenylalanine at position 330 with leucine. It has been shown that εFZK / αFVH, which is an activity ratio derived by dividing the enzyme activity for oxycarbonyllysine (hereinafter referred to as εFZK) by the enzyme activity for αFVH, decreases from 0.95 to 0.025.

  However, εFZK, which is used for the evaluation of the substrate specificity of the modified ketoamine oxidase in the above document, is a compound that is considerably different in terms of molecular weight and structure from εFK actually produced in the step of treating glycated hemoglobin with protease. It is. For this reason, it is difficult to say that the reactivity to εFK, which can be a causative substance of actual measurement errors, has decreased due to the decreased reactivity to εFZK. Moreover, there is no description that the reduction | decrease of the reactivity with respect to (epsilon) FK was confirmed using the modified ketoamine oxidase in the said literature.

  In addition, a modified fructosyl amino acid oxidase newly imparted with reactivity to αFVH by introducing an amino acid substitution into a fructosyl amino acid oxidase derived from Aspergillus nidulans A89 and modifying the substrate specificity Has been reported (see Patent Document 11). For example, a fructosyl amino acid oxidase derived from Aspergillus nidulans A89 can be renewed by substituting serine at position 59 with glycine and lysine at position 65 with glycine, or substituting lysine at position 109 with glutamine. It is shown that the enzyme activity for αFVH is imparted. However, there is no description that the amino acid substitution contributes to a reduction in reactivity to εFK.

  That is, examples of amadoriase including natural type or mutant type amadoriase, which has a low εFK / αFVH and / or a low εFK / αFV, which is an activity ratio derived by dividing the enzyme activity for εFK by the enzyme activity for αFVH, However, there is still a need for an amadoriase that has a sufficiently low reactivity to εFK and that can realize highly accurate measurement of HbA1c.

  In order to actually measure HbA1c in a blood sample using various amadoriases, a reagent composition for measurement in which amadoriase and various required components are prescribed is prepared, and this is performed in a predetermined method. Use to measure. For the purpose of obtaining a highly accurate measurement value, in addition to efforts to improve the properties of the enzyme itself, for example, a reagent composition for measurement that is as difficult to be affected as possible by εFK, which can cause a measurement error. It is also important to develop a measurement method that is not easily affected by εFK or the like.

  For example, as an attempt to reduce the influence of εFK and the like, a method of erasing an influence substance such as εFK using another enzyme prior to measuring αFVH by amadoriase is disclosed (for example, Patent Document 17). reference). By reducing the amount of the influential substance in advance by this method, the accuracy of the measurement value can be expected to improve. However, this method has a problem that an additional pre-processing such as an erasing operation is required, and the measuring operation is complicated. Another problem is that the measurement time is increased by the amount of time required for the work associated with the erasing process, such as the time required for erasing and the time for stopping the erasing reaction. Furthermore, the fact that the reagent formulation suitable for erasure, measurement, and storage must be established taking into account the reactivity and stability of the erasure enzyme can be a major limitation in the study of formulation.

  As another attempt to reduce the influence of εFK and the like, a method for measuring αFVH by acting amadoriase under weakly acidic pH conditions such as pH 5.5 and pH 6.5 has been reported (Patent Document 2). reference). By causing the measurement reaction to be performed in such a weakly acidic pH range, the effect of εFK on the measured value is reduced as compared with the case where the measurement reaction is performed in a neutral to weakly alkaline pH range of pH 7 to 8. Is disclosed. However, the optimum reaction pH of the amadoriase disclosed in this document is 8.0 (see Non-Patent Document 1), and the reactivity of this enzyme in a weakly acidic pH region such as pH 5.5, pH 6.5, etc. Is not enough. That is, in this method, in order to avoid the influence of εFK on the measured value, there is a problem that the reagent composition must be assembled so as to be used under conditions where the performance of the enzyme cannot be sufficiently exhibited. As described above, by combining the reagent composition at a pH that deviates from the optimum pH of the enzyme reaction, there is a problem that the amount of the enzyme to be used must be increased.

  That is, in the method for measuring glycated hemoglobin using amadoriase, there is a demand for further improvement that can obtain a measurement value that is simpler, quicker, and highly accurate. Further, in the reagent composition for measuring glycated hemoglobin, there is a demand for further improvement in which usage conditions are less restricted and the performance of the prescribed enzyme can be maximized.

International Publication No. 2004/104203 International Publication No. 2005/49857 JP 2001-95598 A Japanese Patent Publication No. 05-33997 JP-A-11-127895 International Publication No. 97/13872 JP2011-229526A JP 2003-235585 A JP 2004-275013 A JP 2004-275063 A JP 2010-35469 A JP 2010-57474 A International Publication No. 2010/41715 International Publication No. 2010/41419 International Publication No. 2011/15325 JP 2010-233502 A International Publication No. 2007/10950

Biochem. Biophys. Res. Commun. 311, 104-11, 2003 Biotechnol. Bioeng. 106, 358-66, 2010 J. et al. Biosci. Bioeng. 102, 241-3, 2006 Appl. Microbiol. Biotechnol. 74, 813-9, 2007 Eur. J. et al. Biochem. 242, 499-505, 1996 Mar. Biotechnol. 6, 625-32, 2004 Biosci. Biotechnol. Biochem. 66, 1256-61, 2002 Biosci. Biotechnol. Biochem. 66, 2323-29, 2002 Biotechnol. Letters 27, 27-32, 2005 J. et al. Biol. Chem. 279, 27613-20, 2004

  The problem to be solved by the present invention is to provide a method for measuring glycated hemoglobin using amadoriase, which is simpler, quicker and obtains a highly accurate measurement value, and there are restrictions on the use conditions used for that purpose. It is an object of the present invention to provide a reagent composition for measuring glycated hemoglobin that can sufficiently exhibit the performance of a prescribed enzyme.

  As a result of intensive studies in order to solve the above problems, the inventors of the present invention have a remarkable reactivity to εFK with respect to the reactivity to αFVH in a wide pH range from a weakly acidic region to a weakly alkaline region. When a reagent composition for measuring glycated hemoglobin formulated with a reduced amadoriase is used, it is possible to eliminate the influence substances by pretreatment in a wide pH range including the pH most suitable for the reaction of the amadoriase. The inventors have found that a highly accurate measurement value can be obtained without performing a process, and completed the present invention.

That is, the present invention relates to the following.
(1) A reagent composition for measuring glycated hemoglobin comprising a protease and amadoriase, wherein the amadoriase has the following properties (a) and / or (b):
(A) In the range from pH 5.5 to 8.0, the reactivity ratio to ε-fructosyl lysine when the reactivity to α-fructosyl valylhistidine is 1 is 0.05 or less. .
(B) In the range from pH 5.5 to 8.0, the influence of ε-fructosyl lysine coexisting in the sample on the measured value of α-fructosyl valyl histidine is 15% or less.
(2) The reagent composition for measuring glycated hemoglobin according to the above (1), which is formulated so that the measurement reaction with amadoriase is performed at pH 5.5 to 8.0.
(3) In the above (1) or (2), the amadoriase is an amadoriase having a substitution and / or deletion of an amino acid residue shown in the following (c) or (d) in the amino acid sequence shown in SEQ ID NO: 1. The reagent composition for measuring glycated hemoglobin as described.
(C) Substitution of glutamic acid at position 98 to alanine, substitution of serine at position 154 to asparagine, substitution of valine at position 259 to cysteine (d) Substitution of glutamic acid at position 98 to alanine, substitution of glycine at position 103 Substitution of histidine, substitution of serine at position 154, substitution of asparagine at position 263, substitution of glycine at position 263, substitution of leucine at position 265, substitution of phenylalanine at position 435, proline at position 435, lysine at position 436, and leucine at position 437 Deletion (4) Amadoriase has the substitution and / or deletion of the amino acid residue shown in (c) or (d) above in the amino acid sequence shown in SEQ ID NO: 1, and shown in (c) or (d) Amateurs having amino acid sequences in which one or several amino acid deletions, insertions, additions and / or substitutions have been made at positions other than Glycated hemoglobin measurement reagent composition according to according to (1) or (2) is a lyase.
(5) A method for measuring glycated hemoglobin using the reagent composition for measuring glycated hemoglobin according to any one of (1) to (4).
(6) A kit for measuring glycated hemoglobin comprising the reagent composition for measuring glycated hemoglobin according to any one of (1) to (4) above.

  According to the present invention, it is possible to provide a reagent composition for measuring glycated hemoglobin in which the influence on the measurement by εFK is reduced in a wide pH range from a weakly acidic region to a weakly alkaline region. Since the reagent composition for measuring glycated hemoglobin of the present invention can be suitably used in a wide pH range including the pH most suitable for the reaction of amadoriase, a formulation that maximizes the performance of the enzyme is possible. Moreover, since there are few restrictions of pH in prescription, it is easy to perform flexible prescription according to the use and the kind of compounding component. In particular, in the past, in the vicinity of a pH suitable for the measurement reaction, a pretreatment elimination step was necessary to avoid the influence of εFK on the measurement, whereas according to the present invention, the optimum pH for the measurement reaction was obtained. The reagent composition for measuring glycated hemoglobin of the present invention and the method for measuring glycated hemoglobin using the same are industrially useful in that, even when used, a highly accurate measurement value can be obtained without performing steps such as elimination of the affected substance. Very useful.

It is a figure which shows reaction optimal pH of Amadoriase CFP-T9-98A / 154N / 259C. It is a figure which shows reaction optimal pH of Amadoriase CFP-T9-98A / 103H / 154N / 263M / 265F / (DELTA) PTS1.

  Hereinafter, the present invention will be described in detail.

(Glycated protein)
The glycated protein in the present invention refers to a non-enzymatically glycated protein. Glycated proteins exist both inside and outside the body, and examples of existing in vivo include glycated hemoglobin and glycated albumin in blood. Among glycated hemoglobins, the β-chain amino terminal valine of hemoglobin is glycated. Glycated hemoglobin is particularly referred to as HbA1c. Examples that exist outside the living body include foods and drinks such as liquid seasonings in which proteins, peptides and sugars coexist, and infusions.

(Amadoriase)
Amadoriase is also known as ketoamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, fructosyl valyl histidine oxidase, fructosyl amine oxidase, and oxidizes iminodiacetic acid or its derivative (Amadori compound) in the presence of oxygen. An enzyme that catalyzes a reaction that produces glyoxylic acid or α-ketoaldehyde, an amino acid or peptide, and hydrogen peroxide. Amadoriase is widely distributed in nature and can be obtained by searching for microorganisms, enzymes of animal or plant origin. The microorganism can be obtained from, for example, filamentous fungi, yeast, or bacteria.

  The amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention is a modified version of amadoriase having a modified substrate specificity, which is produced based on the amadoriase derived from the genus Coniochaeta having the amino acid sequence shown in SEQ ID NO: 1. . Examples of such mutants include high sequence identity with SEQ ID NO: 1 (eg, 75% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95). %, More preferably 97% or more, most preferably 99% or more), and in the amino acid sequence of SEQ ID NO: 1, one to several amino acids are altered or mutated or deleted And amadoriase having a substituted, added and / or inserted amino acid sequence. As long as the conditions relating to the amino acid sequence described in the claims are satisfied, for example, Eupenicillium, Pyrenoceta, Arsulinium, Carbraria, Neocosmospola, Cryptococcus, Feosferia, Aspergillus, Emerycera , Urocradium, Penicillium, Fusarium, Acaetomera, Acaetomium, Sierravia, Caetmium, Gerasinospora, Microascus, Leptoferia, Offiobolus, Preospola, Coniochetidium, Pichia, Corynebacterium It may be produced based on amadoriase derived from other species such as Agrobacterium and Arthrobacter.

Prior to the present application, the applicant found a plurality of amino acid mutation sites that are effective in modifying the substrate specificity at pH 7.0 as single mutations, and this finding was found in International Publication No. 2012/18094 after the priority date of the present application. It is published as. However, the effect of modifying the substrate specificity by amino acid substitution described in the above document has been confirmed only at pH 7.0, and it is optimal for the reaction of amadoriase when such amino acid substitution is accumulated. It was not clear whether it was possible to produce an amadoriase capable of obtaining a highly accurate measurement value in a wide pH range including pH, without performing a process such as separately erasing the affected substance by pretreatment. Thus, as a result of further trial and error, the inventors have found that the modified Amadoriase having the amino acid residue substitution and / or deletion shown in (c) or (d) below in the amino acid sequence shown in SEQ ID NO: 1 is reacted. In the wide pH range including the optimum pH, it has been found that it is an amadoriase capable of obtaining highly accurate measurement values without performing steps such as elimination of the influence substance by pretreatment.
(C) Substitution of glutamic acid at position 98 to alanine, substitution of serine at position 154 to asparagine, substitution of valine at position 259 to cysteine (d) Substitution of glutamic acid at position 98 to alanine, substitution of glycine at position 103 Substitution of histidine, substitution of serine at position 154, substitution of asparagine at position 263, substitution of glycine at position 263, substitution of leucine at position 265, substitution of phenylalanine at position 435, proline at position 435, lysine at position 436, and leucine at position 437 Deletion Furthermore, the amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention has the amino acid substitution and / or deletion described above, and the amino acid sequence shown in SEQ ID NO: 1 has been substituted and / or deleted. 90% or more, more preferably 95% or more, relative to the amino acid sequence of the portion excluding amino acids. More preferably, it includes an amadoriase variant that has an amino acid sequence identity of 97% or more, particularly preferably 99% or more, has an amadoriase activity, and has a modified substrate specificity.
Furthermore, the amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention has substitution and / or deletion of the amino acid residue shown in (c) or (d) above in the amino acid sequence shown in SEQ ID NO: 1. Amadoriase variants having amino acid sequences in which one or several amino acid deletions, insertions, additions and / or substitutions have been made at positions other than the positions shown in (c) or (d).

  In the above amino acid substitution, the amino acid position represents the position in the amino acid sequence of the amadoriase derived from the genus Coniocaeta shown in SEQ ID NO: 1. However, in the amino acid sequence of the amadoriase derived from another species, The amino acid at the position corresponding to the position in the amino acid sequence to be substituted is substituted. The meaning of “corresponding position” will be described later.

(Amino acid sequence homology)
Amino acid sequence homology can be calculated using GENETYX-Mac (manufactured by Software Development), such as maximum matching and search homology programs, or DNASIS Pro (manufactured by Hitachi Software), such as maximum matching and multiple alignment programs. .

(Acquisition of gene encoding amadoriase)
In order to obtain the gene of the present invention encoding these amadoriases (hereinafter also simply referred to as “amadoriase gene”), generally used gene cloning methods are used. For example, chromosomal DNA or mRNA can be extracted from microbial cells having the ability to produce amadoriase and various cells by a conventional method, for example, a method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using mRNA as a template. A chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA thus obtained.

Subsequently, based on the amino acid sequence of the amadoriase, a suitable probe DNA is synthesized, and using this, a method for selecting the amadoriase gene from a chromosomal DNA or cDNA library, or a suitable primer DNA based on the amino acid sequence. And amplifying DNA containing the desired gene fragment encoding amadoriase by an appropriate polymerase chain reaction (PCR method) such as 5′RACE method or 3′RACE method, Can be ligated to obtain a DNA containing the full length of the target amadoriase gene.
A preferred example of the gene encoding the amadoriase thus obtained is the Amadoriase gene derived from the genus Coniocaeta (see Japanese Patent Application Laid-Open No. 2003-235585).

  It is preferable in handling that these amadoriase genes are linked to various vectors as usual. For example, from a recombinant plasmid pKK223-3-CFP (see Japanese Patent Application Laid-Open No. 2003-235585) containing DNA encoding an amadoriase gene derived from Coniochaeta sp. NISL 9330, GenElute Plasmid Miniprep Kit (manufactured by Sigma-Alcrich) Can be used to extract and purify DNA encoding the amadoriase gene.

(vector)
The vector that can be used to produce amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention is not limited to the above-mentioned plasmid, and other persons skilled in the art such as bacteriophage, cosmid, etc. Any known vector can be used. Specifically, for example, pBluescript II SK + (Stratagene) is preferable.

(Amadoriase gene mutation treatment)
The mutation process of the amadoriase gene can be performed by any known method depending on the intended mutant form. That is, a wide variety of methods such as a method of contacting and acting an amadoriase gene or a recombinant DNA incorporating the gene and a mutagen agent; an ultraviolet irradiation method; a genetic engineering method; or a method using a protein engineering method. Can be used.

  As a method of making full use of a protein engineering technique, a technique generally known as Site-Specific Mutagenesis can be used. For example, Kramer method (Nucleic Acids Res., 12, 9441 (1984): Methods Enzymol., 154, 350 (1987): Gene, 37, 73 (1985)), Eckstein method (Nucleic Acids Res., 13, 87). 1985): Nucleic Acids Res., 13, 8765 (1985): Nucleic Acids Res, 14, 9679 (1986)), Kunkel method (Proc. Natl. Acid. Sci. USA, 82, 488). ): Methods Enzymol., 154, 367 (1987)).

  In addition, a technique known as a general PCR method can be used (see Technique, 1, 11 (1989)). In addition to the above gene modification method, a desired modified amadoriase gene can also be directly synthesized by an organic synthesis method or an enzyme synthesis method.

  When the DNA base sequence of the amadoriase gene obtained by the above method is determined or confirmed, it can be performed by using, for example, a multicapillary DNA analysis system Applied Biosystems 3130xl Genetic Analyzer (manufactured by Life Technologies).

(Transformation / Transduction)
The amadoriase gene obtained as described above is incorporated into a vector such as a bacteriophage, a cosmid, or a plasmid used for transformation of prokaryotic cells or eukaryotic cells by a conventional method, and a host corresponding to each vector is selected by a conventional method. Can be transformed or transduced. For example, using a microorganism belonging to the genus Escherichia as a host, for example, the obtained recombinant DNA, for example, Escherichia coli K-12 strain, preferably Escherichia coli JM109 strain, Escherichia coli DH5α strain (both manufactured by Takara Bio Inc.), etc. Transform or transduce them to obtain the respective strain.

(Identification of the position corresponding to the amino acid)
The “position corresponding to an amino acid” refers to the position in the amino acid sequence of an amadoriase derived from another species corresponding to the amino acid at a specific position in the amino acid sequence of the amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1.
As a method for specifying “position corresponding to amino acid”, amino acid sequences are compared using a known algorithm such as Lippmann-Person method, and the maximum homology to conserved amino acid residues present in the amino acid sequences of each amadoriase It can be done by giving sex. By aligning the amino acid sequences of the amadoriases in this way, it is possible to determine the positions of the homologous amino acid residues in the sequence of each amadoriase sequence regardless of insertions or deletions in the amino acid sequences. The homologous position is considered to exist at the same position in the three-dimensional structure, and it can be estimated that the homologous position has a similar effect on the specific function of the target amadoriase.

In the present invention, the “position corresponding to glutamic acid at position 98 of the amino acid sequence described in SEQ ID NO: 1” refers to the confirmed amino acid sequence of Amadoriase and the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. When compared, it means an amino acid corresponding to glutamic acid at position 98 of the amadoriase of SEQ ID NO: 1. Thereby, the amino acid sequence can be aligned and specified by the above-mentioned method of specifying “the amino acid at the corresponding position”.
That is, amadoriase derived from Eupenicillium terrenum, fructosyl amino acid oxidase derived from Penicillium janthinellum, serine at position 98, ketoamine oxidase derived from Pyrenochaeta sp., Ketoamine oxidase derived from Curvularia clavata, fructosyl amino acid oxidase derived from Cryptococcus neoformans, Phaeosphaeria nodorum Fructosyl peptide oxidase derived from Ulocladium sp. Fructosyl amino acid oxidase derived from Ulocladium sp. 98th alanine, ketoamine oxidase derived from Arthrinium sp., Ketoamine oxidase derived from Neocosmospora vasinfecta In the amino acid oxidase, fructosyl peptide oxidase derived from Emericella nidulans, it is serine at position 97.

In the present invention, the “position corresponding to glycine at position 103 of the amino acid sequence described in SEQ ID NO: 1” refers to the confirmed amino acid sequence of Amadoriase and the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. When compared, it means the amino acid corresponding to glycine at position 103 of the amadoriase of SEQ ID NO: 1. Thereby, the amino acid sequence can be aligned and specified by the above-mentioned method of specifying “the amino acid at the corresponding position”.
That is, amadoriase derived from E. terrenum, ketoamine oxidase derived from Pyrenochaeta sp., Ketoamine oxidase derived from Arthrinium sp., Ketoamine oxidase derived from C. clavata, ketoamine oxidase derived from N. vasinfecta, derived from Ulocladium sp. Fructosyl amino acid oxidase, glycine at position 103 for fructosyl amino acid oxidase from P. janthinellum, serine at position 103 for fructosyl amino acid oxidase from C. neoformans, and aspartic acid at position 103 for fructosyl peptide oxidase from P. nodorum In the fructosyl amino acid oxidase derived from A. nidulans and the fructosyl peptide oxidase derived from E. nidulans, it is glycine at position 102.

Furthermore, “the position corresponding to serine at position 154 in the amino acid sequence described in SEQ ID NO: 1” means that the confirmed amino acid sequence of Amadoriase is compared with the amino acid sequence of the Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means the amino acid corresponding to serine at position 154 of the amadoriase of SEQ ID NO: 1. This can also be specified by aligning amino acid sequences by the above method.
Namely, E. terrenum-derived amadoriase, P. janthinellum-derived fructosyl amino acid oxidase, cysteine at position 154, Pyrenochaeta sp.-derived ketoamine oxidase, Arthrinium sp.-derived ketoamine oxidase, C. clavata-derived ketoamine oxidase , Ketoamine oxidase from N. vasinfecta, fructosyl amino acid oxidase from C. neoformans, serine at position 154 in fructosyl amino acid oxidase from Ulocladium sp., Serine at position 152 in fructosyl peptide oxidase from P. nodorum, In the fructosyl amino acid oxidase derived from A. nidulans and the fructosyl peptide oxidase derived from E. nidulans, it is a cysteine at position 153.

Furthermore, “the position corresponding to valine at position 259 of the amino acid sequence described in SEQ ID NO: 1” means that the confirmed amino acid sequence of Amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means the amino acid corresponding to valine at position 259 of the amadoriase of SEQ ID NO: 1. This can also be specified by aligning amino acid sequences by the above method.
That is, amadoriase derived from E. terrenum, ketoamine oxidase derived from Arthrinium sp., Ketoamine oxidase derived from N. vasinfecta, fructosyl amino acid oxidase derived from C. neoformans, fructosyl amino acid oxidase derived from A. nidulans, E. nidulans Fructosyl peptide oxidase derived from Ulocladium sp., Fructosyl amino acid oxidase derived from P. janthinellum, valine at position 259, ketoamine oxidase derived from Pyrenochaeta sp., Ketoamine oxidase derived from C. clavata In valine at position 257, fructosyl peptide oxidase derived from P. nodorum, it is valine at position 255.

Furthermore, in the present invention, the “position corresponding to glycine at position 263 of the amino acid sequence described in SEQ ID NO: 1” refers to the confirmed amino acid sequence of Amadoriase and the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. When compared, it means the amino acid corresponding to glycine at position 263 of the amadoriase of SEQ ID NO: 1. Thereby, the amino acid sequence can be aligned and specified by the above-mentioned method of specifying “the amino acid at the corresponding position”.
That is, amadoriase derived from E. terrenum, ketoamine oxidase derived from Arthrinium sp., Ketoamine oxidase derived from N. vasinfecta, fructosyl amino acid oxidase derived from A. nidulans, fructosyl peptide oxidase derived from E. nidulans, P. janthinellum In the fructosyl amino acid oxidase derived from glycine at position 263, ketoamine oxidase derived from Pyrenochaeta sp., Ketoamine oxidase derived from C. clavata, fructosyl amino acid oxidase derived from Ulocladium sp. Glycine derived from C. neoformans In fructosyl amino acid oxidase, it is serine at position 263, and in fructosyl peptide oxidase derived from P. nodorum, it is glycine at position 259.

Furthermore, in the present invention, “position corresponding to leucine at position 265 of the amino acid sequence described in SEQ ID NO: 1” refers to the confirmed amino acid sequence of Amadoriase and the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. When compared, it means the amino acid corresponding to leucine at position 265 of the amadoriase of SEQ ID NO: 1. Thereby, the amino acid sequence can be aligned and specified by the above-mentioned method of specifying “the amino acid at the corresponding position”.
That is, in E. terrenum-derived amadoriase, A. nidulans-derived fructosyl amino acid oxidase, E. nidulans-derived fructosyl peptide oxidase, and P. janthinellum-derived fructosyl amino acid oxidase, tyrosine at position 265, keto derived from Pyrenochaeta sp. Leucine at position 263 for amine oxidase, ketoamine oxidase from Arthrinium sp., Fructosyl amino acid oxidase from C. neoformans, leucine at position 265, ketoamine oxidase from C. clavata, fructosyl amino acid oxidase from Ulocladium sp. Is valine at position 263, ketoamine oxidase from N. vasinfecta is valine at position 265, and fructosyl peptide oxidase from P. nodorum is valine at position 261.

(Position corresponding to the carboxyl-terminal deletion)
“Position corresponding to proline at position 435 of the amino acid sequence described in SEQ ID NO: 1, lysine at position 436, and leucine at position 437” refers to a peroxisome translocation signal of 3 amino acid residues from the carboxyl terminus of the amino acid sequence described in SEQ ID NO: 1 (Peroxisome Targeting Signal 1; PTS1) sequence. In any amadoriase whose amino acid sequence has been determined, the “position corresponding to the proline at position 435 of the amino acid sequence described in SEQ ID NO: 1 and the position corresponding to lysine at position 436 and leucine at position 437” is from the carboxyl terminus that can be estimated as the PTS1 sequence. Of 3 amino acid residues.

  That is, Amadoriase derived from E. terrenum comprises alanine at position 435, histidine at position 436 and leucine at position 437, and ketoamine oxidase from Pyrenochaeta sp. Comprises alanine at position 438, lysine at position 439 and leucine at position 440, and Arthrinium sp. derived ketoamine oxidase consists of histidine at position 450, lysine at position 451 and leucine at position 452, and ketoamine oxidase from C. clavata consists of serine at position 438, lysine at position 439 and leucine at position 440, P The fructosyl peptide oxidase derived from nodorum comprises an alanine at position 435, an asparagine at position 436, and a leucine at position 437, and a fructosyl amino acid oxidase derived from A. nidulans and an 436th position in the fructosyl peptide oxidase derived from E. nidulans. Fructosyl amino acid oxidase derived from Ulocladium sp. Is composed of alanine at position 439, lysine at position 440 and leucine at position 441, and is derived from P. janthinellum. The three amino acids at the carboxyl terminal are alanine at position 435, lysine at position 436, and leucine at position 437.

  In eukaryotes, a PTS1 sequence, which is a signal sequence for transporting a protein to the peroxisome and is a motif composed of three amino acids at the carboxyl terminus, is known. As a well-known publicly known PTS1 motif, there is a motif composed of (proline / serine / alanine / cysteine)-(lysine / histidine / arginine / asparagine)-(leucine / methionine) sequence (for example, FEBS J., 272). 2362 (2005), Plant Cell Physiol., 38, 759 (1997), Eur. J. Cell Biol., 71, 248 (1996)).

(Production of amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention)
In order to produce the amadoriase using a strain having the ability to produce amadoriase with improved substrate specificity obtained as described above, this strain may be cultured by a normal solid culture method, It is preferable to employ the liquid culture method as much as possible.

  Examples of the medium for culturing the above strain include, for example, yeast extract, tryptone, peptone, meat extract, corn steep liquor or one or more nitrogen sources such as soybean or wheat bran leachate, sodium chloride, dihydrogen phosphate. Add one or more inorganic salts such as potassium, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate, and add sugar raw materials, vitamins, etc. as necessary. Used.

  In addition, the production amount of the target enzyme can be obtained by adding a substrate on which the amadoriase can act or a similar compound such as glycated amino acid, glycated peptide, glycated protein degradation product, or glycated protein such as glycated hemoglobin or glycated albumin. Can be improved.

  The initial pH of the medium is suitably adjusted to pH 7-9. The culture is performed at a culture temperature of 20 to 42 ° C., preferably at a culture temperature of about 37 ° C. for 4 to 24 hours, more preferably at a culture temperature of about 37 ° C. for 8 to 16 hours. It is preferably carried out by culturing or the like.

  To collect amadoriase from the culture after completion of the culture, it can be obtained using a normal enzyme collecting means. For example, the cells are subjected to ultrasonic disruption treatment, grinding treatment, or the like, or the enzyme is extracted using a lytic enzyme such as lysozyme, or shaken or left in the presence of toluene or the like for lysis. This enzyme can be discharged out of the cells. Then, this solution is filtered, centrifuged, etc. to remove the solid part, and if necessary, nucleic acid is removed with streptomycin sulfate, protamine sulfate, manganese sulfate or the like, and then ammonium sulfate, alcohol, acetone or the like is added thereto. The fraction is collected and the precipitate is collected to obtain a crude enzyme of amadoriase.

  In order to further obtain an amadoriase purified enzyme preparation from the crude enzyme of the above amadoriase, for example, gel filtration method using Sephadex, Superdex or Ultrogel, ion exchange carrier, hydrophobic carrier, adsorption elution method using hydroxyapatite, Select electrophoresis method using polyacrylamide gel, sedimentation method such as sucrose density gradient centrifugation, affinity chromatography method, fractionation method using molecular sieve membrane or hollow fiber membrane, etc. By doing so, a purified amadoriase enzyme preparation can be obtained. In this way, an amadoriase with reduced reactivity to the desired εFK can be obtained.

(Reduction in reactivity to εFK in amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention)
The amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention obtained by the above means has a substrate specificity compared to that before modification as a result of mutation in the amino acid sequence due to genetic modification or the like. Is improved. Specifically, “reactivity ratio to εFK when the reactivity to αFVH is 1” or “reactivity to εFK when the reactivity to αFV is 1” as compared to that before the modification. The ratio is reduced. Or, as compared to those before modification, “the ratio of reactivity to εFK when the reactivity to αFVH is 1” and “the ratio of reactivity to εFK when the reactivity to αFV is 1” Are all reduced.

  In the measurement of glycated hemoglobin, a high reactivity with εFK can cause measurement errors. Therefore, the lower the ratio of reactivity with εFK, the better. Specifically, in the amadoriase of the present invention, εFK / αFVH indicating the reactivity ratio to εFK with respect to the reactivity to αFVH is preferably a reactivity ratio to εFK when the reactivity to αFVH is 1. It is desirable to show 0.05 or less, more preferably 0.04 or less, further preferably 0.03 or less, and most preferably 0.01 or less.

  In addition, in the amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention, εFK / αFV, which indicates the ratio of reactivity to εFK to the reactivity to αFV, is the reactivity to εFK when the reactivity to αFV is 1. The ratio is preferably 0.03 or less, more preferably 0.01 or less, and most preferably 0.

  The ratio of reactivity to εFK to the reactivity to αFVH or the ratio of reactivity to εFK to the reactivity to αFV was measured under any conditions using a known amadoriase measurement method. Can be compared. For example, the ratio of the reactivity to εFK to the reactivity to αFVH can be calculated by dividing the activity measured by adding 1 mM εFK at an arbitrary pH by the activity measured by adding 1 mM αFVH. It is possible to calculate and compare this before and after modification. In addition, for example, by determining the activity measured by adding 1 mM εFK at an arbitrary pH divided by the activity measured by adding 1 mM αFV, the reactivity of εFK to the reactivity to αFV can be obtained. The ratio can be calculated and compared with the one before modification and the one after modification.

  An example of an amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention, which has improved substrate specificity compared to that before modification, is, for example, Escherichia coli JM109 (pKK223-3-CFP-T9-98A / 154N / 259C) and Amadoriase produced by E. coli JM109 (pKK223-3-CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1). Such an amadoriase with improved substrate specificity is well reduced in the degree to which εFK is measured as noise, and is glycated from αFVH, which is a glycated amino acid derived from the β-chain amino terminus of HbA1c, or from the β-chain amino terminus of HbA1c. Since only αFV, which is an amino acid, can be measured, highly accurate measurement can be performed, which is very advantageous for industrial use.

(Reduction of the effect of εFK on the measured value of αFVH in amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention)
The amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention has a substrate specificity improved as compared with that before modification due to a mutation in its amino acid sequence. As a result, αFVH is measured using the amadoriase of the present invention. In this case, the influence on the measured value of αFVH when εFK that can cause a measurement error coexists can be reduced. Specifically, the influence of εFK coexisting in the sample on the measured value of αFVH is, for example, measured with (αFVH + εFK) mixed sample when the change in absorbance is 100% when a sample containing only αFVH is measured. The ratio of the change in absorbance is preferably 85% to 115%, more preferably 95% to 105%, still more preferably 97% to 103%, and most preferably 98% to 102%. It is preferable.

  In addition, when the ratio of the absorbance change when measuring the (αFVH + εFK) mixed sample when the absorbance change when measuring a sample containing only αFVH is 100% is within the range of 85% to 115%, εFK Is expressed as being 15% or less, and when it is within 95% or more and 105% or less, it is expressed that εFK has an effect on the measured value of αFVH being 5% or less. When it falls within the range of not less than 100% and not more than 103%, the influence of εFK on the measured value of αFVH is expressed as 3% or less. Further, when it falls within the range of 98% to 102%, it is expressed that the influence of εFK on the measured value of αFVH is 2% or less.

(Reduction in the effect of εFK on the measured value of αFV in amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention)
The amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention has a mutation in its amino acid sequence and improved substrate specificity compared to that before modification. As a result, αFV is measured using the amadoriase of the present invention. In this case, the influence on the measured value of αFV when εFK that can cause a measurement error coexists can be reduced. Specifically, the influence of εFK coexisting in the sample on the measured value of αFV is, for example, (αFV + εFK) mixed sample when the amount of change in absorbance is 100% when a sample containing only αFV is measured. It is preferable that the ratio of the change in absorbance at that time is preferably 95% to 105%, more preferably 97% to 103%, and still more preferably 98% to 102%.

  In addition, when the ratio of the absorbance change when measuring the (αFV + εFK) mixed sample when the absorbance change amount when measuring a sample containing only αFV is 100% is within 95% to 105%, εFK Is expressed as being 5% or less, and if it is within 97% to 103%, it is expressed that the effect of εFK on the measured value of αFV is 3% or less. Further, when it falls within the range of 98% to 102%, it is expressed that the influence of εFK on the measured value of αFVH is 2% or less.

(PH range where the effect of improving substrate specificity in amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention is exhibited)
The amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention has a substrate specificity improved as a result of mutation in its amino acid sequence, and the reagent for measuring glycated hemoglobin of the present invention. When αFVH is measured using an amadoriase that can be used in the composition, it is possible to reduce the influence on the measured value of αFVH when εFK that can cause a measurement error coexists. It is also a feature of the present invention that the above-described effect of amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention is exhibited in a wide pH range. Specifically, the effect of improving the substrate specificity of amadoriase that can be used in the reagent composition for measuring glycated hemoglobin of the present invention and the effect of reducing the influence on the measured value of αFVH when εFK coexists are from pH 5.5. It is demonstrated over a wide range of 8.0. This is very advantageous in practice. In order to obtain the above-mentioned effects, the conventional amadoriase has a restriction that it must be used in a pH range that is considerably away from the pH optimum for the reaction of the enzyme. For example, in some cases, the reaction must be carried out on the acidic side even though the optimum reaction pH is alkaline. However, in the reagent for measuring glycated hemoglobin of the present invention, the reaction can be performed under a pH condition closer to the optimum reaction pH or under the same pH condition as the optimum reaction pH, and the enzyme performance was sufficiently exhibited. Measurement can be performed in the state. Alternatively, when the reaction pH must be intentionally changed for the purpose of conforming to the measurement reagent components other than the enzyme and various conditions of the measurement equipment, the pH range is less likely to be constrained. Thus, the effects of the present invention can be exhibited.

  Regarding the liquid composition in the composition for measuring glycated hemoglobin of the present invention, a glycated hemoglobin degrading reagent containing a protease, a glycated peptide / amino acid measuring reagent containing an amadoriase for oxidatively deglycosylating the produced glycated peptide or glycated amino acid, Peroxidase (POD) for measuring the amount of hydrogen peroxide by measuring the color intensity by coloring the reducing agent by oxidation-reduction reaction using hydrogen peroxide generated by the oxidative deglycation reaction of, and by oxidation What is necessary is just to combine suitably the reagent for hydrogen peroxide amount measurement containing the reducing agent which develops color. In addition, these reagents can be provided as liquid products and liquid frozen products or freeze-dried products.

  As a protease for degrading glycated hemoglobin blended in the composition for measuring glycated hemoglobin of the present invention, a protease that liberates glycated peptide / amino acid from glycated hemoglobin, most preferably αFVH from the β-chain amino terminus of glycated hemoglobin. You can choose. For example, neutral protease derived from the genus Bacillus can be mentioned. The specific conditions for reacting the protease are not particularly limited as long as the desired glycated peptide / amino acid can be prepared, and are suitably suitable depending on the concentration and type of the sample and the type of protease. It may be adopted after considering the conditions.

Regarding the pH range of the composition for measuring glycated hemoglobin of the present invention, the pH is selected so that the reaction proceeds efficiently in consideration of the optimum pH of the amadoriase to be used, and then the amount of the enzyme that acts on the glycated amino acid is determined. do it. For example, when CFP-T9-98A / 154N / 259C is used, pH is 5.5 to 8.0, preferably pH 6.5 to 8.0, which is a pH region showing 90% or more of the maximum activity. Preferably, pH 7.0 to 7.5, which is a pH range showing 95% or more of the maximum activity, can be selected.
A major feature of the composition for measuring glycated hemoglobin of the present invention is that the influence of εFK can be satisfactorily avoided when used over a wide pH range that has not been realized so far. Of course, the influence of εFK can be satisfactorily avoided even in a weakly acidic region outside the region showing activity, but a pH range suitable for the reaction of amadoriase unless there are other constraints imposed by the prescribed ingredients and the type of measuring instrument It is more desirable to be able to prescribe in the vicinity.

  Moreover, the addition concentration of amadoriase should just be a density | concentration which can fully detect glycated peptide / amino acid in the reaction liquid used, when adding CFP-T9-98A / 154N / 259C, for example, 0.1- 500 U / ml, preferably 0.5 to 200 U / ml, most preferably 1.0 to 100 U / ml. However, the concentration is not particularly limited, and can be determined as appropriate according to the reaction conditions, the type and state of the sample, the procedure of the experimenter, the type of reagent used, and the like.

  In addition, the type and concentration of the buffer solution used for adjusting the reagent pH to the target range are not particularly limited as long as the buffer solution exhibits a buffering ability in a desired pH range. Buffers exhibiting buffer capacity in the range of 5 to pH 8.0 include dipotassium hydrogen phosphate / disodium-potassium dihydrogen phosphate / sodium buffer, MES-NaOH buffer, Bis-Tris-HCl buffer, PIPES-NaOH buffer solution, MOPS-NaOH buffer solution, HEPES-NaOH buffer solution, Tris-HCl buffer solution and the like can be mentioned.

The peroxidase (POD) blended in the composition for measuring glycated hemoglobin of the present invention is not particularly limited as long as it is a POD capable of developing a reducing agent by an oxidation-reduction reaction using hydrogen peroxide. For example, POD derived from horseradish and microorganisms can be selected, and may be adopted after considering suitable conditions according to the concentration and type of the sample and the concentration and type of protease.
Any protease that liberates αFVH can be selected. For example, neutral protease derived from the genus Bacillus can be mentioned. The specific conditions for reacting the protease are not particularly limited as long as the desired saccharified amine can be prepared. Appropriate conditions are appropriately selected according to the concentration and type of the sample and the type of POD. It may be adopted after consideration.

  The reducing agent for detecting hydrogen peroxide blended in the composition for measuring glycated hemoglobin of the present invention is not particularly limited as long as it develops color by the catalytic action of POD in the presence of hydrogen peroxide. For example, a tender reagent that produces a dye by subjecting a coupler such as 4-aminoantipyrine (4-AA) to an oxidative condensation reaction of a phenol-based, aniline-based or toluidine-based chromogen, and Leuco dyes that oxidize directly in the presence of POD can be used. Examples of the coupler include 4-aminoantipyrine, aminoantipyrine derivatives, vanillin diamine sulfonic acid, methylbenzthiazolinone hydrazone (MBTH), and sulfonated methylbenzthiazolinone hydrazone (SMBTH). The Trinder reagent is not limited. For example, phenol such as N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline (TOOS) and derivatives thereof can be used. Although it does not specifically limit as a leuco pigment | dye, For example, a triphenylmethane derivative, a phenothiazine derivative, a diphenylamine derivative, etc. can be used.

(Glycated hemoglobin measurement method, measurement kit, pretreatment, post-treatment process)
Glycated hemoglobin can be measured by replacing the composition for measuring glycated hemoglobin of the present invention with a conventional composition for measuring glycated hemoglobin. Moreover, the kit for measuring glycated hemoglobin of the present invention can be produced by replacing the composition for measuring glycated hemoglobin in the conventional kit for measuring glycated hemoglobin with the composition for measuring glycated hemoglobin of the present invention. Since the composition for measuring glycated hemoglobin of the present invention can be suitably used for a wider range of pH, for example, the pH in a region where it has not been possible to prioritize avoiding the influence of εFK. It is also possible to formulate a kit prescription.
In the measurement method and measurement kit, any pretreatment and posttreatment conventionally used can be included. When the composition for measuring glycated hemoglobin according to the present invention is used, it is expected that the step of eliminating the influence substance that has been necessary in the past is unnecessary by adjusting the conditions. In particular, in the neutral to weak alkaline pH range, when the elimination process, which was indispensable for obtaining highly accurate measurement values, is no longer necessary, the reagent manufacturing cost, measurement work, and measurement time are all greatly improved. It is extremely useful in that efficient measurement and kit production are realized.

(Method for measuring amadoriase activity)
Various methods can be used as a method for measuring the activity of amadoriase. As an example, a method for measuring amadoriase activity used in the present invention will be described below.

  Examples of the method for measuring the enzyme activity of amadoriase in the present invention include a method for measuring the amount of hydrogen peroxide produced by the reaction of the enzyme and a method for measuring the amount of oxygen consumed by the enzyme reaction. As an example, a method for measuring the amount of hydrogen peroxide will be described below.

In the present invention, αFV, αFVH, or εFK is used as a substrate for the measurement of amadoriase activity unless otherwise specified. The enzyme titer is defined as 1 U for the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute when measured using αFV, αFVH, or εFK as a substrate.
As glycated amino acids such as αFV and glycated peptides such as αFVH, for example, those synthesized and purified based on the method of Sakagami et al. can be used (see JP 2001-95598 A).

A. : Reagent (Reagent 1): 5 U / ml peroxidase, 0.49 mM 4-aminoantipyrine 0.1 M phosphate buffer 5.0 kU peroxidase (Kikkoman), 100 mg 4-aminoantipyrine (Japanese) Dissolved in 0.1M potassium phosphate buffer (pH 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0) and adjusted to a volume of 1000 ml To do.
(Reagent 2): A 15 mM TOOS solution 500 mg of TOOS (manufactured by Dojindo Laboratories) is dissolved in ion-exchanged water, and the volume is adjusted to 100 ml.
(Reagent 3): αFVH solution (300 mM; final concentration 10 mM)
Dissolve 1250 mg of αFVH in ion-exchanged water, and make up to 10 ml.
(Reagent 4): Substrate solution (30 mM; final concentration 1 mM)
125 mg of αFVH or 92 mg of εFK or 84 mg of αFV is dissolved in ion-exchanged water, and the volume is adjusted to 10 ml.

B: Optimum pH measurement method for reaction 2.7 ml of reagent 1, 100 μl of reagent 2, and 100 μl of enzyme solution are mixed and pre-warmed at 37 ° C. for 5 minutes. Thereafter, 100 μl of Reagent 3 was added and mixed well, and the time-dependent change in absorbance at 555 nm was observed with a spectrophotometer (U-3010A, manufactured by Hitachi High-Technologies Corporation), and the amount of change per minute in absorbance at 555 nm ( ΔAs) was measured. For the control solution, the amount of change per minute (ΔA0) in absorbance at 555 nm was measured in the same manner as described above except that 100 μl of ion-exchanged water was added instead of 100 μl of reagent 3. The number of micromoles of hydrogen peroxide produced per minute at 37 ° C. was defined as the activity unit (U) in the enzyme solution, and the calculation was performed according to the following formula.

Activity (U / ml) = {(ΔAs−ΔA0) × 3.0 × df} ÷ (39.2 × 0.5 × 0.1)

ΔAs: Change in absorbance per minute of reaction solution ΔA0: Change in absorbance per minute of control solution
39.2: millimolar extinction coefficient (mM ^-1 · cm ^-1 ) of quinoneimine dyes produced by the reaction
0.5: 1 mol of quinoneimine dye produced by 1 mol of hydrogen peroxide
df: Dilution factor

C: Substrate specificity measurement method 2.7 ml of reagent 1, 100 μl of reagent 2, and 100 μl of enzyme solution are mixed and pre-warmed at 37 ° C. for 5 minutes. Thereafter, 100 μl of Reagent 4 was added and mixed well, and the change with time in absorbance at 555 nm was observed with a spectrophotometer (U-3010A, manufactured by Hitachi High-Technologies Corporation), and the amount of change per minute in absorbance at 555 nm ( ΔAs) was measured. For the control solution, the amount of change per minute (ΔA0) in absorbance at 555 nm was measured in the same manner as described above except that 100 μl of ion-exchanged water was added instead of 100 μl of reagent 4. The number of micromoles of hydrogen peroxide produced per minute at 37 ° C. was defined as the activity unit (U) in the enzyme solution, and was calculated according to the following formula.

Activity (U / ml) = {(ΔAs−ΔA0) × 3.0 × df} ÷ (39.2 × 0.5 × 0.1)

ΔAs: Change in absorbance per minute of reaction solution ΔA0: Change in absorbance per minute of control solution
39.2: millimolar extinction coefficient (mM ^-1 · cm ^-1 ) of quinoneimine dyes produced by the reaction
0.5: 1 mol of quinoneimine dye produced by 1 mol of hydrogen peroxide
df: Dilution factor

(Amadoriase reaction optimum pH)
The optimum pH of the reaction of amadoriase is determined from the relative activity at each pH when the activity at the pH showing the maximum activity is 100% as a result of measuring the amadoriase activity at each pH in accordance with B: Optimal pH measurement method for reaction. . The optimum reaction pH in the present invention refers to a pH range in which the relative activity is 95% or more, more preferably 100%.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.
The relationship between CFP, CFP-T7 and CFP-T9 described in the examples is shown in Table 1.

Preparation of Amadoriase derived from Coniochaeta genus (1) Preparation of recombinant plasmid pKK223-3-CFP-T9 DNA SEQ ID NO: 1 is derived from the genus Coniochaeta into which a heat-stabilized mutation (G184D, F265L, N272D, H302R, H388Y) has been introduced. It is an amino acid sequence of amadoriase and is encoded by the gene of SEQ ID NO: 2 (see International Publication No. 2007/1225779).
Escherichia coli JM109 (pKK223-3-CFP-T9) strain (see International Publication No. 2007/12579) having a recombinant plasmid of the Amadoriase gene derived from the genus Coniochaeta (SEQ ID NO: 2) was added to 3 ml of LB-amp medium [1% ( w / v) bactotryptone, 0.5% (w / v) peptone, 0.5% (w / v) NaCl, 50 μg / ml ampicillin], and cultured with shaking at 37 ° C. for 16 hours. A culture was obtained.
The culture was collected by centrifugation at 10,000 × g for 1 minute to obtain bacterial cells. From this bacterial cell, the recombinant plasmid pKK223-3-CFP-T9 was extracted and purified using GenElute Plasmid Mini-Prep Kit (manufactured by Sigma-Aldrich), and 2.5 μg of the recombinant plasmid pKK223-3 was purified. -CFP-T9 DNA was obtained.

(2) Site-specific modification operation of recombinant plasmid pKK223-3-CFP-T9 DNA Glutamic acid at position 98 in the amino acid sequence described in SEQ ID NO: 1 is alanine, serine at position 154 is asparagine, and valine at position 259 is In order to substitute for cysteine, using the recombinant plasmid pKK223-3-CFP-T9 DNA as a template, the synthetic oligonucleotides of SEQ ID NOS: 6 and 7, KOD-Plus- (manufactured by Toyobo Co., Ltd.) were used under the following conditions: PCR reaction was performed.
That is, 5 μl of 10 × KOD-Plus-buffer, 5 μl of dNTPs mixed solution prepared so that each dNTP is 2 mM, 2 μl of 25 mM MgSO ₄ solution, and 50 ng of pKK223-3-CFP-T9 DNA as a template Each of the above synthetic oligonucleotides was added at 15 pmol and 1 unit of KOD-Plus-, and the total volume was adjusted to 50 μl with sterilized water. The prepared reaction solution was incubated at 94 ° C. for 2 minutes using a thermal cycler (Eppendorf), followed by “94 ° C., 15 seconds” — “50 ° C., 30 seconds” — “68 ° C., 6 minutes”. This cycle was repeated 30 times.

  A part of the reaction solution was electrophoresed on a 1.0% agarose gel, and it was confirmed that about 6,000 bp of DNA was specifically amplified. The DNA thus obtained was treated with a restriction enzyme DpnI (manufactured by NEW ENGLAND BIOLABS), and the remaining template DNA was cleaved. Then, Escherichia coli JM109 was transformed and developed on an LB-amp agar medium. The grown colonies were inoculated into LB-amp medium and cultured with shaking, and plasmid DNA was isolated by the same method as in (1) above. The base sequence of DNA encoding amadoriase in the plasmid was determined using a multicapillary DNA analysis system Applied Biosystems 3130xl Genetic Analyzer (manufactured by Life Technologies), and as a result, the 98th position of the amino acid sequence described in SEQ ID NO: 1 was determined. A recombinant plasmid (pKK223-3-CFP-T9-98A) encoding a modified amadoriase in which glutamic acid was replaced with alanine was obtained.

  In the same manner as described above, using pKK223-3-CFP-T9-98A DNA as a template and using the synthetic oligonucleotides of SEQ ID NOS: 8 and 9, glutamic acid at position 98 is replaced with alanine and serine at position 154 is replaced with asparagine. A recombinant plasmid (pKK223-3-CFP-T9-98A / 154N) encoding the modified amadoriase thus obtained was obtained.

  Further, in the same manner as described above, using pKK223-3-CFP-T9-98A / 154N DNA as a template and using the synthetic oligonucleotides of SEQ ID NOS: 10 and 11, glutamic acid at position 98 is alanine and serine at position 154 is A recombinant plasmid (pKK223-3-CFP-T9-98A / 154N / 259C) encoding a modified amadoriase in which valine at position 259 was replaced with cysteine was obtained in asparagine.

(3) Production of CFP-T9-98A / 154N / 259C E. coli JM109 (pKK223-3-CFP-T9) obtained by transforming E. coli with pKK223-3-CFP-T9-98A / 154N / 259C -98A / 154N / 259C) was inoculated into 40 ml of LB-amp medium supplemented with 0.1 mM IPTG and cultured at 25 ° C. for 16 hours. After washing each of the obtained cultured cells with 20 mM HEPES-NaOH buffer (pH 7.0), the cells were suspended in the same buffer and subjected to ultrasonic crushing treatment at 20,000 × g for 10 minutes. Centrifugation was performed to prepare 8 ml of a crude enzyme solution.
(4) Purification of CFP-T9-98A / 154N / 259C

  The prepared crude enzyme solution was adsorbed on 4 ml of Q Sepharose Fast Flow resin (manufactured by GE Healthcare) equilibrated with 20 mM HEPES-NaOH buffer (pH 7.0), and then the resin was washed with 80 ml of the same buffer. Subsequently, the protein adsorbed on the resin was eluted with 20 mM HEPES-NaOH buffer (pH 7.0) containing 100 mM NaCl, and a fraction showing amadoriase activity was collected.

  The obtained fraction showing amadoriase activity was concentrated with Amicon Ultra-15, 30K NMWL (manufactured by Millipore). Thereafter, it is applied to HiLoad 26/60 Superdex 200 pg (manufactured by GE Healthcare) equilibrated with 20 mM HEPES-NaOH buffer (pH 7.0) containing 150 mM NaCl, eluted with the same buffer, and fractions showing amadoriase activity. Were collected, and purified samples of wild type and modified amadoriase were obtained. The obtained purified sample was confirmed to be purified to a single band by analysis by SDS-PAGE.

(5) Preparation of Recombinant Plasmid pKK223-3-CFP-T7 DNA SEQ ID NO: 3 is the amino acid sequence of the Amadoriase derived from the genus Coniochaeta into which the heat-stability improving mutation (N272D, H302R, H388Y) has been introduced, SEQ ID NO: 4 (See International Publication No. 2007/12579).
Escherichia coli JM109 (pKK223-3-CFP-T7) strain (see International Publication No. 2007/12579) having a recombinant plasmid of the Amadoriase gene (SEQ ID NO: 4) derived from the genus Coniochaeta is obtained in the same manner as in the above (1). The cells were cultured and collected to obtain microbial cells. From this bacterial cell, the recombinant plasmid pKK223-3-CFP-T7 was extracted and purified using GenElute Plasmid Mini-Prep Kit (Sigma-Aldrich), and 2.5 μg of the recombinant plasmid pKK223-3 was purified. -CFP-T7 DNA was obtained.

(6) Site-specific modification operation of recombinant plasmid pKK223-3-CFP-T7 DNA In the amino acid sequence shown in SEQ ID NO: 1, glutamic acid at position 98 is alanine, glycine at position 103 is histidine, and serine at position 154 is Asparagine modified valine at position 259, glycine at position 263, methionine at position 263, leucine at position 265 with phenylalanine, and proline at position 435, lysine at position 436, leucine at position 437 In order to prepare amadoriase, the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template and the synthetic oligonucleotides of SEQ ID NOS: 12 and 13 and KOD-Plus- (manufactured by Toyobo Co., Ltd.) were used. PCR reaction, transformation of Escherichia coli JM109 strain and growth Chromatography was carried out sequencing of DNA encoding the amadoriase of plasmid DNA to be retained. As a result, a recombinant plasmid (pKK223-3-CFP-T7-184D) encoding a modified amadoriase in which glycine at position 184 of the amino acid sequence described in SEQ ID NO: 3 was substituted with aspartic acid was obtained.

  In the same manner as described above, using pKK223-3-CFP-T7-184D DNA as a template and using synthetic oligonucleotides of SEQ ID NOS: 14 and 15, glycine at position 184 of the amino acid sequence shown in SEQ ID NO: 3 was converted to aspartic acid. A recombinant plasmid (pKK223-3-CFP-T7-184D / ΔPTS1) encoding a modified amadoriase which was substituted and lacked proline at position 435, lysine at position 436, and leucine at position 437 was obtained.

  Subsequently, in the same manner as described above, the pKK223-3-CFP-T7-184D / ΔPTS1 DNA was used as a template, and the synthetic oligonucleotides of SEQ ID NOS: 6 and 7 were used. A recombinant plasmid (pKK223-3-3) encoding a modified amadoriase in which glutamic acid is replaced with alanine, glycine at position 184 is replaced with aspartic acid, and proline at position 435, lysine at position 436, and leucine at position 437 are deleted. CFP-T7-98A / 184D / ΔPTS1) was obtained.

  Subsequently, using the synthetic oligonucleotides of SEQ ID NOS: 16 and 17 using pKK223-3-CFP-T7-98A / 184D / ΔPTS1 DNA as a template in the same manner as described above, the amino acid sequence 98 of SEQ ID NO: 3 was used. The modified glutamate is substituted with alanine, glycine at position 103 is replaced with histidine, glycine at position 184 is replaced with aspartic acid, and proline at position 435, lysine at position 436, and leucine at position 437 are encoded. A recombinant plasmid (pKK223-3-CFP-T7-98A / 103H / 184D / ΔPTS1) was obtained.

  Subsequently, in the same manner as described above, pKK223-3-CFP-T7-98A / 103H / 184D / ΔPTS1 DNA was used as a template, and the synthetic oligonucleotides of SEQ ID NOs: 8 and 9 were used, and the amino acid sequence described in SEQ ID NO: 3 The glutamic acid at position 98 is alanine, glycine at position 103 is replaced with histidine, serine at position 154 is replaced with asparagine, glycine at position 184 is replaced with aspartic acid, and proline at position 435, lysine at position 436, and position 437. A recombinant plasmid (pKK223-3-CFP-T7-98A / 103H / 154N / 184D / ΔPTS1) encoding a modified amadoriase lacking leucine was obtained.

  Further, in the same manner as above, pKK223-3-CFP-T7-98A / 103H / 154N / 184D / ΔPTS1 DNA was used as a template, and the synthetic oligonucleotides of SEQ ID NOs: 18 and 19 were used, and the amino acids described in SEQ ID NO: 3 In the sequence, glutamic acid at position 98 is alanine, glycine at position 103 is histidine, serine at position 154 is asparagine, glycine at position 184 is aspartic acid, glycine at position 263 is replaced with methionine, and proline at position 435 A modified amadoriase lacking lysine at position 436, leucine at position 437, ie, glutamic acid at position 98 in the amino acid sequence shown in SEQ ID NO: 1 is alanine, glycine at position 103 is histidine, and serine at position 154 is asparagine. 263th glycine is methionine and 265th Recombinant plasmid (pKK223-3-CFP-T9-98A / 103H / 154N) encoding a modified amadoriase in which leucine is substituted with phenylalanine and proline at position 435, lysine at position 436, and leucine at position 437 are deleted / 263M / 265F / ΔPTS1).

(7) Production of CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 Obtained by transforming Escherichia coli with pKK223-3-CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 The obtained Escherichia coli JM109 (pKK223-3-CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1) strain was inoculated into 40 ml of LB-amp medium supplemented with 0.1 mM IPTG, and the strain was incubated at 25 ° C. for 16 minutes. Incubate for hours. After washing each of the obtained cultured cells with 20 mM HEPES-NaOH buffer (pH 7.0), the cells were suspended in the same buffer and subjected to ultrasonic crushing treatment at 20,000 × g for 10 minutes. Centrifugation was performed to prepare 8 ml of a crude enzyme solution.

(8) Purification of CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 A crude enzyme solution containing the prepared CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 is described in (4) above. Purification according to the method gave a purified sample of CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1. The obtained purified sample was confirmed to be purified to a single band by analysis by SDS-PAGE.

(9) Measurement of optimum reaction pH of CFP-T9-98A / 154N / 259C and CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 CFP-T9-98A / 154N / 259C and CFP-T9 Using a purified preparation of −98A / 103H / 154N / 263M / 265F / ΔPTS1, the optimum reaction pH was measured by the method shown in the above B: optimum reaction pH measurement method. As shown in FIGS. 1 and 2, the optimum reaction pH of CFP-T9-98A / 154N / 259C and CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 were both 7.5.

(10) Measurement of εFK / αFVH and εFK / αFV Using the above-described purified samples of CFP-T9-98A / 154N / 259C and CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1, C: Enzyme activity against αFVH, αFV and εFK was measured by the method shown in the substrate specificity measurement method. Subsequently, the ratio of reactivity to αFVH to the reactivity to εFK at each pH (εFK / αFVH) and the ratio of reactivity to αFV to the reactivity to εFK (εFK / αFV) were calculated. As a comparative example, the same measurement was performed using a purified sample of CFP (SEQ ID NO: 5) (manufactured by Kikkoman Biochemifa). The calculation results of εFK / αFVH are shown in Table 2, and the calculation results of εFK / αFV are shown in Table 3.

  In CFP-T9-98A / 154N / 259C and CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1, both εFK / αFVH and εFK / αFV are extremely low compared to CFP as a comparative example. Value. In the comparative example, although the value was relatively low in the weakly acidic pH region, it was confirmed that the value was remarkably increased as it changed from the neutral region to the weakly alkaline region, and was easily affected by εFK. On the other hand, in the mutant of the present invention, εFK / αFVH is 0 to 0.03 and εFK / αFV is 0 to 0.01 in a wide pH range ranging from 5.5 to 8.0. It was confirmed that it was difficult to be affected by.

Measurement of fructosyl amino acid samples and fructosyl peptide samples

  Using the CFP obtained as described above and the purified sample of CFP-T9-98A / 154N / 259C, the following samples were measured. The activity values of CFP and CFP-T9-98A / 154N / 259C were adjusted to pH 8.0 using αFV at a final concentration of 5 mM as a substrate according to the method described in “C: Substrate specificity measurement method” described above. Determined using (Reagent 1).

(11) Preparation of fructosyl amino acid sample and fructosyl peptide sample Various fructosyls with a substrate solution of 0.5 mM were obtained by diluting the αFVH, εFK or αFV solution described in (Reagent 4) 60 times with ion-exchanged water. Samples of amino acids or fructosyl peptides were prepared.

(12) Measurement of fructosyl amino acid sample and fructosyl peptide sample (reagent 5)
0.86U / ml CFP or CFP-T9-98A / 154N / 259C
0.93 U / ml peroxidase (Kikkoman)
0.46 mM 4-aminoantipyrine (manufactured by Wako Pure Chemical Industries, Ltd.)
0.52 mM TOOS (Dojindo Laboratories)
93 mM phosphate buffer (pH 6.0, 7.0, 7.5, or 8.0)

To the 34 μl sample prepared in (11) above, 986 μl of (Reagent 5), which had been pre-warmed at 37 ° C. for 5 minutes, was added to start the reaction, and the wavelength after reaction at 550 nm for 5 minutes at 555 nm Was measured with a spectrophotometer U-3010A. The absorbance change amount when each sample was measured was calculated according to the following formula using the absorbance at a wavelength of 555 nm (reagent blank) measured in the same manner using ion-exchanged water instead of the sample as a control.

Absorbance change = ΔAes−Ae0
ΔAes: Absorbance after 5 minutes from the start of reaction ΔAe0: Absorbance after 5 minutes from the start of reaction when a control solution is added

Using each obtained absorbance change amount, ΔAεFK / ΔAαFVH corresponding to the ratio of the absorbance change amount of εFK when the absorbance change amount of αFVH was set to 1 was calculated. The results are shown in Table 4. Table 5 shows ΔAεFK / ΔAαFV corresponding to the ratio of the change in absorbance of εFK, where the amount of change in absorbance of αFV calculated in the same manner is 1.

  As is clear from Tables 4 and 5, ΔAεFK / ΔAαFVH and ΔAεFK / ΔAαFV measured using a reagent containing CFP-T9-98A / 154N / 259C were low at all pHs. In particular, even at pH 7.5, which is the optimum pH of this enzyme, ΔAεFK / ΔAαFVH and ΔAεFK / ΔAαFV of CFP-T9-98A / 154N / 259C all have the lowest values in the case of CFP as a comparative example. The obtained pH was lower than ΔAεFK / ΔAαFVH and ΔAεFK / ΔAαFV at 6.0, indicating that the influence of εFK could be greatly reduced over a wide pH range.

  In addition, the following samples were measured using the purified samples of CFP and CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 obtained as described above. The activity values of CFP and CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 were adjusted to pH 7.0 using αFVH at a final concentration of 5 mM as a substrate according to the method described in C: Substrate specificity measurement method. It was determined using the adjusted (Reagent 1).

(13) Preparation of fructosyl amino acid sample and fructosyl peptide sample Various fructosyls with a substrate solution of 0.3 mM were prepared by diluting the αFVH, εFK or αFV solution described in (Reagent 4) 100 times with ion-exchanged water. Samples of amino acids or fructosyl peptides were prepared.

(14) Measurement of fructosyl amino acid sample and fructosyl peptide sample (reagent 6)
0.72 U / ml CFP, or 0.96 U / ml CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1
5.4 U / ml peroxidase (Kikkoman)
0.53 mM 4-aminoantipyrine (Wako Pure Chemical Industries, Ltd.)
0.6 mM TOOS (manufactured by Dojin Chemical Laboratory)
108 mM phosphate buffer (pH 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0)

The reaction was started by adding 35 μl of the sample prepared in (13) above to 175 μl of (Reagent 6) that had been pre-warmed at 37 ° C. for 5 minutes, and at a wavelength of 545 nm after the reaction at 37 ° C. for 5 minutes. Absorbance was measured with an automatic analyzer Bio Majesty JCA-BM1650 (JEOL). The absorbance change amount when each sample was measured was calculated according to the following equation using the absorbance at a wavelength of 545 nm (reagent blank) measured in the same manner using ion-exchanged water instead of the sample as a control.

Absorbance change = ΔAes−Ae0
ΔAes: Absorbance after 5 minutes from the start of reaction ΔAe0: Absorbance after 5 minutes from the start of reaction when a control solution is added

Using each obtained absorbance change amount, ΔAεFK / ΔAαFVH corresponding to the ratio of the absorbance change amount of εFK when the absorbance change amount of αFVH was set to 1 was calculated. The results are shown in Table 6. Table 7 shows ΔAεFK / ΔAαFV corresponding to the ratio of the amount of change in absorbance of εFK when the amount of change in absorbance of αFV calculated in the same manner is 1.

  As is apparent from Tables 6 and 7, ΔAεFK / ΔAαFVH and ΔAεFK / ΔAαFV measured using reagents including CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 were low at all pH values. . In particular, even at pH 7.5, which is the optimum pH of the present enzyme, ΔAεFK / ΔAαFVH and ΔAεFK / ΔAαFV of CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 are all the comparative examples of CFP. In this case, the lowest values were obtained at ΔAεFK / ΔAαFVH and ΔAεFK / ΔAαFV at 6.0, the pH at which the lowest values were obtained, and the influence of εFK was greatly reduced over a wide pH range. I understood.

Measurement of fructosyl amino acid / fructosyl peptide sample coexisting with εFK

(15) Preparation of a fructosyl amino acid or fructosyl peptide sample containing εFK The αFV or εFK solution described in (Reagent 4) is mixed and diluted with ion-exchanged water, thereby allowing 0.5 μm each of αFV and εFK. Mixed samples containing (αFV + εFK) were prepared. Similarly, the αFVH or εFK solution described in (Reagent 4) was mixed and diluted with ion-exchanged water to prepare a mixed sample containing 0.5 mM each of αFVH and εFK (αFVH + εFK).

(16) Measurement of fructosyl amino acid and fructosyl peptide samples containing εFK To 34 μl of the sample prepared in the above (15), 986 μl of (Reagent 4) that had been pre-heated at 37 ° C. for 5 minutes was added. The reaction was started, and the absorbance at a wavelength of 555 nm after the reaction at 37 ° C. for 5 minutes was measured using a spectrophotometer U-3010A. The absorbance change amount when each sample was measured was calculated according to the following formula using the absorbance at a wavelength of 555 nm (reagent blank) measured in the same manner using ion-exchanged water instead of the sample as a control.

Absorbance change = ΔAes−Ae0
ΔAes: Absorbance after 5 minutes from the start of reaction ΔAe0: Absorbance after 5 minutes from the start of reaction when a control solution is added

Using the obtained absorbance change amounts, the ratio ΔA (αFVH + εFK) / (AFVH + εFK) / (AFVH + εFK) / (AFVH + εFK) / (AFVH + εFK) when measuring the mixed sample when the absorbance change amount when measuring a sample containing only αFVH is 1. ΔAαFVH was calculated. The results are shown in Table 8. Similarly, the ratio ΔA (αFV + εFK) / ΔAαFV of the change in absorbance when the (αFV + εFK) mixed sample was measured when the absorbance change when measuring a sample containing only αFV calculated in the same manner as 1 is shown in Table 9 It was shown to.

  As is apparent from Tables 8 and 9, ΔA (αFVH + εFK) / ΔAαFVH and ΔA (αFV + εFK) / ΔAαFV measured using a reagent containing CFP-T9-98A / 154N / 259C were low at all pH values. . Especially when CFP-T9-98A / 154N / 259C is used even at pH 7.5, which is the optimum pH of this enzyme, αFVH and αFV can be accurately quantified without being affected by coexisting εFK. I understood. In addition, the same measurement was attempted under conditions on the acidic side of pH 5.5. However, pH 5.0 is outside the pH range where the quinoneimine dye produced by the oxidative condensation reaction of TOOS and 4-AA exhibits coloration. In the formulations described in the examples, accurate quantification based on the amount of change in absorbance was impossible. From the above, it was confirmed that the reagent composition of the present invention can be suitably used for measurement in a wide range of pH 5.5 to 8.0, and the influence of εFK on the measured value of αFVH over this pH range is 15%. It was the following. In particular, the influence of εFK on the measured value of αFVH over pH 5.5 to 7.5 was 5% or less, and it was confirmed that it can be suitably used in such a wide range measurement.

(17) Preparation of fructosyl amino acid and fructosyl peptide sample containing εFK The αFV or εFK solution described in (Reagent 4) is mixed, and diluted with ion-exchanged water, so that αFV and εFK are each 0.3 mM. Mixed samples containing (αFV + εFK) were prepared. Similarly, the αFVH or εFK solution described in (Reagent 4) was mixed and diluted with ion-exchanged water to prepare a mixed sample containing 0.3 mM each of αFVH and εFK (αFVH + εFK).

(18) Measurement of fructosyl amino acid and fructosyl peptide samples containing εFK 35 μl of the sample prepared in (18) above was added to 175 μl of (Reagent 6) that had been pre-warmed at 37 ° C. for 5 minutes. The reaction was started, and the absorbance at a wavelength of 545 nm after the reaction at 37 ° C. for 5 minutes was measured using an automatic analyzer Bio Majesty JCA-BM1650. The absorbance change amount when each sample was measured was calculated according to the following equation using the absorbance at a wavelength of 545 nm (reagent blank) measured in the same manner using ion-exchanged water instead of the sample as a control.

Absorbance change = ΔAes−Ae0
ΔAes: Absorbance after 5 minutes from the start of reaction ΔAe0: Absorbance after 5 minutes from the start of reaction when a control solution is added

Using the obtained absorbance change amounts, the ratio ΔA (αFVH + εFK) / (AFVH + εFK) / (AFVH + εFK) / (AFVH + εFK) / (AFVH + εFK) when measuring the mixed sample when the absorbance change amount when measuring a sample containing only αFVH is 1. ΔAαFVH was calculated. The results are shown in Table 10. Similarly, the ratio ΔA (αFV + εFK) / ΔAαFV of the amount of change in absorbance when the (αFV + εFK) mixed sample is measured when the amount of change in absorbance when measuring a sample containing only αFV calculated in the same manner is 1 is shown in Table 11. It was shown to.

  As is apparent from Tables 9 and 10, ΔA (αFVH + εFK) / ΔAαFVH and ΔA (αFV + εFK) / ΔAαFV measured using a reagent containing CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 are all pH values. The value was low. In particular, even at pH 7.5, which is the optimum pH of the present enzyme, when CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 is used, αFVH and αFVH are not affected by coexisting εFK. It was found that αFV can be accurately quantified. From the above, it was confirmed that the reagent composition of the present invention can be suitably used for measurement in a wide range of pH 5.5 to 8.0, and the influence of εFK on the measured value of αFVH over this pH range is 2%. Hereinafter, the influence of εFK on the measured value of αFV was 3% or less. Moreover, the influence which (epsilon) FK has on the measured value of (alpha) FV over pH 5.5-7.5 is 2% or less, and it was confirmed that it can be used conveniently in such a wide range measurement. In addition, CFP-T9-98A / 103H / 154N / 263M / 265F / ΔPTS1 has improved thermal stability compared to CFP-T9, and has been modified to amadoriase which is superior in terms of stability.

Claims (5)

In the reagent composition for measuring glycated hemoglobin containing protease and amadoriase, the measurement reaction by amadoriase is formulated at pH 5.5 to 8.0, and the amadoriase has the following properties (a) and / or (b): A reagent composition for measuring glycated hemoglobin,
(A) In the range from pH 7.0 to 8.0, the reactivity ratio to ε-fructosyl lysine when the reactivity to α-fructosyl valylhistidine is 1 is 0.05 or less. ,
(B) In the range from pH 7.0 to 8.0, the effect of ε-fructosyl lysine coexisting in the sample on the measured value of α-fructosyl valyl histidine is 15% or less.
Here, the amadoriase is an amadoriase derived from Coniochaeta sp., A ketoamine oxidase derived from E. terrenum, a ketoamine oxidase derived from Arthrinium sp., A ketoamine oxidase derived from N. vasinfecta, a fructosyl amino acid oxidase derived from C. neoformans, Fructosyl amino acid oxidase from A. nidulans, fructosyl peptide oxidase from E. nidulans, fructosyl amino acid oxidase from Ulocladium sp., Fructosyl amino acid oxidase from P. janthinellum, ketoamine oxidase from Pyrenochaeta sp., C Amadoriase having 95% or more amino acid sequence identity with clavata-derived ketoamine oxidase or P. nodorum-derived fructosyl peptide oxidase, and the amino acid residues shown in (c) or (d) below Replacement and A / or amadoriase having a deletion,
(C) glutamic acid at position 98 in the amino acid sequence shown in SEQ ID NO: 1,
Serine at position 98 in amadoriase from Eupenicillium terrenum, Serine at position 98 in fructosyl amino acid oxidase from Penicillium janthinellum,
Glutamic acid at position 98 in ketoamine oxidase from Arthrinium sp.,
Glutamic acid at position 98 in ketoamine oxidase from Neocosmospora vasinfecta,
Serine at position 97 in fructosyl amino acid oxidase from Aspergillus nidulans, or
Serine at position 97 in fructosyl peptide oxidase from Emericella nidulans,
Substitution of alanine with
Serine at position 154 in the amino acid sequence shown in SEQ ID NO: 1,
Cysteine at position 154 in the amadoriase from E. terrenum,
Cysteine at position 154 in fructosyl amino acid oxidase from P. janthinellum,
Serine at position 154 in ketoamine oxidase from Pyrenochaeta sp.
Serine at position 154 in the ketoamine oxidase from Arthrinium sp.
Serine at position 154 in ketoamine oxidase from C. clavata,
Serine at position 154 in the ketoamine oxidase from N. vasinfecta,
Serine at position 154 in fructosyl amino acid oxidase from C. neoformans;
Serine at position 154 in fructosyl amino acid oxidase from Ulocladium sp., Serine at position 152 in fructosyl peptide oxidase from P. nodorum, cysteine at position 153 in fructosyl amino acid oxidase from A. nidulans, or
Substitution of cysteine at position 153 in fructosyl peptide oxidase from E. nidulans with asparagine, and valine at position 259 in the amino acid sequence shown in SEQ ID NO: 1,
Valine at position 259 in amadoriase from E. terrenum,
Valine at position 259 in the ketoamine oxidase from Arthrinium sp.,
Valine at position 259 in the ketoamine oxidase from N. vasinfecta,
Valine at position 259 in fructosyl amino acid oxidase from C. neoformans,
Valine at position 259 in the fructosyl amino acid oxidase from A. nidulans;
Valine at position 259 in fructosyl peptide oxidase from E. nidulans;
Valine at position 259 in fructosyl amino acid oxidase from Ulocladium sp.,
Valine at position 259 in fructosyl amino acid oxidase from P. janthinellum, valine at position 257 in ketoamine oxidase from Pyrenochaeta sp.,
Valine at position 257 in ketoamine oxidase from C. clavata, or
Valine at position 255 in fructosyl peptide oxidase from P. nodorum,
Substitution of cysteine with
(D) glutamic acid at position 98 in the amino acid sequence shown in SEQ ID NO: 1,
Serine at position 98 in amadoriase from Eupenicillium terrenum,
Serine at position 98 in fructosyl amino acid oxidase from Penicillium janthinellum,
Glutamic acid at position 98 in ketoamine oxidase from Arthrinium sp.,
Glutamic acid at position 98 in ketoamine oxidase from Neocosmospora vasinfecta,
Serine at position 97 in fructosyl amino acid oxidase from Aspergillus nidulans, or
Serine at position 97 in fructosyl peptide oxidase from Emericella nidulans,
Substitution of alanine with
Glycine at position 103 in the amino acid sequence shown in SEQ ID NO: 1,
Glycine at position 103 in the amadoriase from E. terrenum,
Glycine at position 103 in ketoamine oxidase from Pyrenochaeta sp.
Glycine at position 103 in ketoamine oxidase from Arthrinium sp.,
Glycine at position 103 in ketoamine oxidase from C. clavata,
Glycine at position 103 in the ketoamine oxidase from N. vasinfecta,
Glycine at position 103 in fructosyl amino acid oxidase from Ulocladium sp., Glycine at position 103 in fructosyl amino acid oxidase from P. janthinellum,
Serine at position 103 in fructosyl amino acid oxidase from C. neoformans,
Aspartic acid at position 103 in fructosyl peptide oxidase from P. nodorum,
Glycine at position 102 in fructosyl amino acid oxidase from A. nidulans, or
Glycine at position 102 in fructosyl peptide oxidase from E. nidulans,
Substitution of histidine,
Serine at position 154 in the amino acid sequence shown in SEQ ID NO: 1,
Cysteine at position 154 in the amadoriase from E. terrenum,
Cysteine at position 154 in fructosyl amino acid oxidase from P. janthinellum,
Serine at position 154 in ketoamine oxidase from Pyrenochaeta sp.
Serine at position 154 in the ketoamine oxidase from Arthrinium sp.
Serine at position 154 in ketoamine oxidase from C. clavata,
Serine at position 154 in the ketoamine oxidase from N. vasinfecta,
Serine at position 154 in fructosyl amino acid oxidase from C. neoformans;
Serine at position 154 in fructosyl amino acid oxidase from Ulocladium sp.
Serine at position 152 in fructosyl peptide oxidase derived from P. nodorum,
Cysteine at position 153 in fructosyl amino acid oxidase from A. nidulans,
Cysteine at position 153 in fructosyl peptide oxidase from E. nidulans,
Of asparagine,
Glycine at position 263 in the amino acid sequence shown in SEQ ID NO: 1
Glycine at position 263 in amadoriase from E. terrenum,
Glycine at position 263 in ketoamine oxidase from Arthrinium sp.
Glycine at position 263 in ketoamine oxidase from N. vasinfecta,
Glycine at position 263 in fructosyl amino acid oxidase from A. nidulans,
Glycine at position 263 in fructosyl peptide oxidase from E. nidulans,
Glycine at position 263 in fructosyl amino acid oxidase from P. janthinellum;
Glycine at position 261 in ketoamine oxidase from Pyrenochaeta sp.
Glycine at position 261 in ketoamine oxidase from C. clavata,
Glycine at position 261 in fructosyl amino acid oxidase from Ulocladium sp., Serine at position 263 in fructosyl amino acid oxidase from C. neoformans, or
Glycine at position 259 in fructosyl peptide oxidase from P. nodorum,
Substitution of methionine,
Leucine at position 265 in the amino acid sequence shown in SEQ ID NO: 1,
Tyrosine at position 265 in amadoriase from E. terrenum,
Tyrosine at position 265 in fructosyl amino acid oxidase from A. nidulans,
Tyrosine at position 265 in fructosyl peptide oxidase from E. nidulans,
Tyrosine at position 265 in fructosyl amino acid oxidase from P. janthinellum,
Leucine at position 263 in ketoamine oxidase from Pyrenochaeta sp.
Leucine at position 265 in the ketoamine oxidase from Arthrinium sp.
Leucine at position 265 in fructosyl amino acid oxidase from C. neoformans,
Valine at position 263 in ketoamine oxidase from C. clavata,
Valine at position 263 in fructosyl amino acid oxidase from Ulocladium sp.
Valine at position 265 in the ketoamine oxidase from N. vasinfecta, or
Valine at position 261 in fructosyl peptide oxidase from P. nodorum,
Substitution of phenylalanine, and proline at position 435 in the amino acid sequence shown in SEQ ID NO: 1, lysine at position 436, and leucine at position 437,
Alanine at position 435, histidine at position 436 and leucine at position 437 in the amadoriase from E. terrenum;
Alanine at position 438, lysine at position 439 and leucine at position 440 in ketoamine oxidase from Pyrenochaeta sp.
Histidine at position 450, lysine at position 451 and leucine at position 452 in the ketoamine oxidase from Arthrinium sp.
Serine at position 438, lysine at position 439 and leucine at position 440 in ketoamine oxidase from C. clavata,
Alanine at position 435, asparagine at position 436 and leucine at position 437 in fructosyl peptide oxidase from P. nodorum,
A fructosyl amino acid oxidase from A. nidulans, alanine at position 436, lysine at position 437 and methionine at position 438 in fructosyl peptide oxidase from E. nidulans;
Alanine at position 439, lysine at position 440 and leucine at position 441 in fructosyl amino acid oxidase from Ulocladium sp., Or
In the fructosyl amino acid oxidase derived from P. janthinellum, the three amino acids at the carboxyl terminal are alanine at position 435, lysine at position 436, and leucine at position 437;
Deletion.   Amadoriase derived from Coniochaeta sp., Ketoamine oxidase derived from E. terrenum, ketoamine oxidase derived from Arthrinium sp., Ketoamine oxidase derived from N. vasinfecta, fructosyl amino acid oxidase derived from C. neoformans, A. fructosyl amino acid oxidase from nidulans, fructosyl peptide oxidase from E. nidulans, fructosyl amino acid oxidase from Ulocladium sp., fructosyl amino acid oxidase from P. janthinellum, ketoamine oxidase from Pyrenochaeta sp., C. clavata The reagent composition for measuring glycated hemoglobin according to claim 1, which has an amino acid sequence identity of 97% or more with a ketoamine oxidase derived from Pt or a fructosyl peptide oxidase derived from P. nodorum.   Amadoriase derived from Coniochaeta sp., Ketoamine oxidase derived from E. terrenum, ketoamine oxidase derived from Arthrinium sp., Ketoamine oxidase derived from N. vasinfecta, fructosyl amino acid oxidase derived from C. neoformans, A. fructosyl amino acid oxidase from nidulans, fructosyl peptide oxidase from E. nidulans, fructosyl amino acid oxidase from Ulocladium sp., fructosyl amino acid oxidase from P. janthinellum, ketoamine oxidase from Pyrenochaeta sp., C. clavata The reagent composition for measuring glycated hemoglobin according to claim 1 or 2, wherein the amino acid sequence identity with the derived ketoamine oxidase or the fructosyl peptide oxidase derived from P. nodorum is 99% or more.   A method for measuring glycated hemoglobin using the reagent composition for measuring glycated hemoglobin according to any one of claims 1 to 3.   A kit for measuring glycated hemoglobin, comprising the reagent composition for measuring glycated hemoglobin according to any one of claims 1 to 3.

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