JP6818718B2 - Modified Amadriase - Google Patents

Description

The present invention relates to a modified amadriase, a gene and recombinant DNA thereof, and a method for producing amadriase having a modified action on glycated amino acids.

A glycated protein is produced by non-enzymatically forming a covalent bond between an aldehyde group of an aldose (a monosaccharide having an aldehyde group and a derivative thereof) such as glucose and an amino group of the protein, and transferring to Amadori. Is. Examples of the amino group of the protein include an amino-terminal α-amino group and an ε-amino group of the lysine residue side chain in the protein. As glycated proteins generated in the living body, glycated hemoglobin in which hemoglobin in blood is saccharified, glycated albumin in which albumin is saccharified, and the like are known.

Among these glycated proteins generated in vivo, glycated hemoglobin (HbA1c) is attracting attention as an important blood glucose control marker for diagnosis and symptom management of diabetic patients in the field of clinical diagnosis of diabetes. Hemoglobin is a protein composed of two α subunits (hereinafter referred to as α chain) and two β subunits (hereinafter referred to as β chain), a total of four subunits, and the amino end of the β chain is glycated. Hemoglobin is called HbA1c. The HbA1c concentration in 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 diabetic symptoms.

As a method for measuring this HbA1c quickly and easily, an enzymatic method using amadriase, that is, α-fructosylvalyl histidine (hereinafter referred to as αFVH) obtained by decomposing HbA1c with a protease or the like and releasing it from the β-chain amino terminus thereof. ), Or a method for quantifying α-fructosylvaline (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 and the quantitativeness is low. Therefore, at present, a method of measuring HbA1c by releasing αFVH from the β chain of HbA1c using a specific protease and quantifying it is the mainstream.

Amadriase catalyzes the reaction of oxidizing imino2acetic acid or its derivatives (also referred to as "Amadori compounds") in the presence of oxygen to produce glyoxylic acid or α-ketoaldehyde, amino acids or peptides, and hydrogen peroxide. To do.
Amadriase has been found in bacteria, yeasts and fungi, but is particularly useful for the measurement of HbA1c, and examples of amadriase having enzymatic activity against αFVH and / or αFV include, for example, the genus Coniochaeta, eupenicilium ( Eupenicillium, Pyrenochaeta, Arthrinium, Curvularia, Neocosmospora, Cryptococcus, Cryptococcus, Feosperia, Feosperia, Feosperia Emericella, Ulocladium, Penicillium, Fusarium, Achaetomiella, Achaetomiella, Achaetomiula, Achaetomium, Sierrabia, Sierrabia, Sierrabia Genus Gelasinospora, genus Microascus, genus Leptosphaeria, genus Opiobolus, genus Pleospora, genus Pleospora, genus Coniochaetidium, genus Coniochaetidium, genus Pionia , Amadriase derived from the genus Agrobacterium and the genus Arthrobacter have been reported (see, for example, Patent Documents 1 and 6 to 16 and Non-Patent Documents 1 to 9). In the above-mentioned reported examples, amadriase may be described by expressions such as ketoamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, fructosylvalyl histidine oxidase, and fructosylamine oxidase depending on the literature. is there.

In the measurement of HbA1c by an enzymatic method, strict substrate specificity is required as a property of amadriase. For example, as described above, when HbA1c is measured by quantifying αFVH released by a specific protease, it is present in the sample in a free state and / or HbA1c using a protease or the like. It is desirable to use amadriase which is difficult to act on glycated amino acids and glycated peptides other than αFVH released in the treatment step. Further, it is known that the amino group at the ε-position of the lysine residue side chain contained in the hemoglobin molecule undergoes saccharification, and the amino group at the ε-position derived from the lysine residue that has undergone this glycation is saccharified. It has been suggested that ε-fructosyllysine (hereinafter referred to as εFK) is released by protease treatment or the like (see Non-Patent Document 10).

In fact, as an HbA1c measurement method currently in practical use, there is a kit that uses whole blood as a sample for measurement, but in whole blood, in addition to αFVH derived from the β-chain amino terminus of HbA1c, , It has been suggested that various glycated amino acids are present (see Patent Document 17). It has been reported that such a phenomenon occurs frequently, for example, especially in patients who have been administered a high-calorie amino acid infusion, and that the same phenomenon can cause a positive error in the measurement of glycated albumin (Non-Patent Documents 11 to 11). See 13). This phenomenon is due to the fact that high-calorie amino acid infusions replenish the body with high concentrations of sugars and amino acids, resulting in the production of free glycated amino acids or glycated peptides in the blood or infusion bags, which are measured. , It is believed that the measured value becomes abnormally high.

Specifically, how much amino acids and sugars are contained in the high-calorie infusion, for example, in the aminotripa (registered trademark) No. 1 infusion (manufactured by Otsuka Pharmaceutical Factory, Ltd.), L-leucine is 31.4 mM. , L-alanine is 26.4 mM, glycine is 23.1 mM, L-valine is 20.1 mM, L-isoleucine is 17.9 mM, and glucose is 521 mM. In addition, L-leucine is 30.9 mM, L-alanine is 29.0 mM, glycine is 22.0 mM, L-valine is 23.0 mM, and L-valine is contained in Unicalic (registered trademark) N infusion solution (manufactured by Terumo). It contains 19.4 mM of isoleucine and 971 mM of glucose. By supplementing the body with an infusion solution containing such various amino acids, various saccharified amino acids such as α-fructosyl leucine (hereinafter referred to as αFL) and α-fructosylalanine (hereinafter referred to as αFA) are contained in the blood. ), Α-fructosylglycine (hereinafter referred to as αFG), αFV, α-fructosyl isoleucine (hereinafter referred to as αFI) and the like can be produced. In measuring HbA1c, since these substances other than αFVH are predicted to be causative substances of measurement error, amadriase having high substrate specificity, which is difficult to act on these glycated amino acids, is strongly desired. ..

As a general technique, in order to modify the substrate specificity of an enzyme, a mutation is added to the DNA encoding the enzyme, a 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, if it is already known that an enzyme having a high amino acid sequence identity has increased substrate specificity by amino acid substitution, it is possible to predict the improvement of substrate specificity based on that information. Is.

In fact, for ketoamine oxidase from Curvularia clavata YH923 and ketoamine oxidase from Neocosmospora vasinfecta 474, substrate specificity for αFVH can be achieved by substituting a few amino acids. An improved modified ketoamine oxidase has been shown (see Patent Document 1). For example, in ketoamine oxidase derived from curbularia clavata YH923, isoleucine at position 58 is replaced with valine, arginine at position 62 is replaced with histidine, and phenylalanine at position 330 is replaced with leucine, thereby ε-fructosyl- (α-benzyl). ΕFZK / αFVH, which is the activity ratio derived by dividing the enzyme activity for oxycarbonyllysine (hereinafter referred to as εFZK) by the enzyme activity for αFVH, is reduced from 0.95 to 0.025, and the enzyme activity for αFV is reduced. It has been shown that αFV / αFVH, which is the activity ratio derived by dividing by the enzyme activity for αFVH, is reduced from 6.3 to 2.2.

However, although the modified ketoamine oxidase disclosed in the above document has reduced αFV / αFVH, it cannot be said that the influence of αFV can be sufficiently avoided. In addition, there is no description that the reduction of reactivity with saccharified amino acids other than εFZK and αFV has been confirmed.

In addition, a modified fructosyl amino acid oxidase that newly imparted reactivity to αFVH by introducing amino acid substitution into fructosyl amino acid oxidase derived from Aspergillus nidulans A89 and modifying the substrate specificity was reported. (See Patent Document 11). For example, in the fructosyl amino acid oxidase derived from Aspergillus nidulans A89, by substituting serine at position 59 with glycine and lysine at position 65 with glycine, or by substituting lysine at position 109 with glutamine, αFVH is newly added. It has been shown that αFV / αFVH is 2.9. However, there is no description that the amino acid substitution contributes to the reduction of reactivity with glycated amino acids, and it cannot be said that the influence of glycated amino acids such as αFV can be sufficiently avoided even in this mutant fructosyl amino acid oxidase.

Although the actual measurement conditions differ from one to another, in known literature, the enzyme activity for εFK and / or the enzyme activity for εFK, which is the activity ratio derived by dividing the enzyme activity for εFK of various amadriase by the enzyme activity for αFVH, is described. There is a disclosure of a modified amadriase with reduced "εFK / αFV", which is the activity ratio derived by dividing by the enzymatic activity for αFV: amadriase from the genus Coniochaeta, fructosyl amino acid oxidase from Aspergillus nidulans, penicillium chrysogenum ΕFK / αFVH and / or Type amadriase has been reported (see Patent Document 18).

For example, by substituting glutamic acid at position 98 of Amadriase from the genus Coniochaeta with alanine, serine at position 154 with asparagine, and valine at position 259 with cysteine, εFK / αFVH was reduced from 0.316 to 0.017. , ΑFV / αFVH have been shown to be reduced from 3.37 to 2.32. Further, a modified amadriase having a low εFK / αFVH has been reported by introducing an amino acid substitution into fructosylvalyl histidine oxidase derived from Phaeosphaeria nodolum and modifying the substrate specificity (see, for example, Patent Document 14).
In addition, by substituting isoleucine at position 58 of fructosylvalyl histidine oxidase derived from Phaeosphaeria nodolum with threonine and phenylalanine at position 282 with tyrosine, εFK / αFVH was reduced from 0.13 to 0.02, and αFV / αFVH. Has been shown to be reduced from 4.9 to 0.5. By further substituting isoleucine at position 58 with threonine, phenylalanine at position 282 for tyrosine, and glutamine at position 110 with phenylalanine, tyrosine, histidine, aspartic acid, or lysine, αFV / αFVH was 4.9 to 0.4. It has been shown to be reduced to.
However, it cannot be said that the influence of αFV is avoided in measuring αFVH by introducing these substitutions.

For example, by substituting threonine for isoleucine at position 58 of fructosylvalyl histidine oxidase derived from Pheasphaeria nodolum described above, αFV / αFVH is reduced from 4.9 to 0.5, but an enzyme having this substitution is used. It has been shown that when 13 μM αFV is coexisted when measuring αFVH (final concentration 26 μM), the measured value is added by about 50% compared to the amount of change in absorbance measured only for αFVH. (See Patent Document 19). Furthermore, there is no description that it contributes to the reduction of reactivity with glycated amino acids other than αFV and εFK.

In addition, in order to actually measure HbA1c in a blood sample using various amadriase, a reagent composition for measurement in which amadriase and various required components are prescribed is prepared, and this is used as a predetermined method. Use in to make measurements. Then, in addition to efforts to improve the properties of the enzyme itself for the purpose of obtaining highly accurate measured values, for example, reagent compositions for measurement that are as insensitive as possible to the effects of εFK, which can be a causative agent of measurement errors, and , ΑFV, εFK, and other saccharified amino acids are also being developed to make measurement methods as insensitive as possible.

For example, as an attempt to reduce the influence of glycated amino acids such as αFV and εFK, a method of eliminating an influential substance such as εFK using another enzyme prior to measuring αFVH by amadriase is disclosed ( For example, see Patent Document 16). By reducing the amount of influential substances in advance by this method, improvement in the accuracy of measured values can be expected. However, this method requires an incidental preprocessing called an erasing operation, and has a problem that the measurement operation is complicated. Another problem is that the measurement time is increased by the time required for the work involved in the erasing process, such as the time required for erasing and the time required for stopping the erasing reaction. Furthermore, the fact that a reagent formulation suitable for erasure, measurement, and storage must be formulated in consideration of the reactivity and stability of the scavenging enzyme can be a major constraint in the examination of the formulation.

As another attempt to reduce the influence of εFK or the like, a method of measuring αFVH by allowing amadriase to act under weakly acidic pH conditions such as pH 5.5 and pH 6.5 has been reported (Patent Document 2). reference). However, there is no mention of avoiding the effects of glycated amino acids other than εFK.

That is, it is affected by glycated amino acids as much as possible by using amadriase having a sufficiently low ratio of reactivity to glycated amino acids other than αFVH to reactivity to αFVH, including natural or mutant amadriase, and such amadriase. Only a few findings have been reported so far for the difficult measurement of αFVH, and there is a demand for an amadriase that has high specificity for αFVH and can realize highly accurate measurement of HbA1c.

International Publication No. 2004/104203 International Publication No. 2005/49857 Japanese Unexamined Patent Publication No. 2001-95598 Special Fair 05-33997 JP-A-11-127895 International Publication No. 97/13872 Japanese Unexamined Patent Publication No. 2011-229526 Japanese Unexamined Patent Publication No. 2003-235585 Japanese Unexamined Patent Publication No. 2004-275013 Japanese Unexamined Patent Publication No. 2004-275603 Japanese Unexamined Patent Publication No. 2010-35469 JP-A-2010-57474 International Publication No. 2010/41715 International Publication No. 2010/41419 International Publication No. 2011/15325 International Publication No. 2007/10950 JP-A-2010-110333 International Publication No. 2012/18094 Japanese Unexamined Patent Publication No. 2010-233501

Biochem. Biophyss. Res. Commun. 311, 104-111, 2003 Biotechnol. Bioeng. 106, 358-66, 2010 J. Biosci. Bioeng. 102, 2413, 2006 Apple. Microbiol. Biotechnol. 74, 815-9, 2007 Euro. J. 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. Biol. Chem. 279, 27613-20, 2004 Inspection and Technology 32, 542-544, 2004 JJCLA 28, 134-138, 2003 Biological sample analysis 27, 97-103, 2004

The problem to be solved by the present invention is an amadriase capable of accurately measuring α-fructosylvalyl histidine even in the presence of glycated amino acids other than α-fructosylvalyl histidine derived from the β-chain amino terminus of glycated hemoglobin. Is to provide.

As a result of intensive research to solve the above problems, the present inventors have replaced specific amino acid residues in amadriase derived from a plurality of microorganisms including the genus Coniocaeta with specific amino acid residues. The present invention has been completed by finding that the problem can be solved.

That is, the present invention is as follows.
(1) The following (i) and (ii):
(I) It has an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 1;
(Ii) Deletion, insertion, addition, and / or substitution of one or several amino acids to glycated amino acids other than α-fructosylvalyl histidine for reactivity to α-fructosylvalyl histidine. Amadriase having the property that the rate of reactivity of is reduced compared to the amadriase before deletion, insertion, addition, and / or substitution.
(2) The following (iii) and (iv):
(Iii) It has an amino acid sequence in which one or several amino acids have been deleted, inserted, added, and / or substituted in the amino acid sequence shown in SEQ ID NO: 1, 30, 33 or 35;
(Iv) Deletion, insertion, addition, and / or substitution of one or several amino acids to glycated amino acids other than α-fructosylvalyl histidine for reactivity to α-fructosylvalyl histidine Amadriase having the property that the rate of reactivity of is reduced compared to the amadriase before deletion, insertion, addition, and / or substitution.
(3) The saccharified amino acids are as follows (a) to (l):
(A) α-fructosyl leucine (b) α-fructosylalanine (c) α-fructosylglycine (d) α-fructosylvaline (e) α-fructosyl isoleucine (f) α-fructosylsleonine ( g) A group consisting of α-fructosylphenylalanine (h) α-fructosylserine (i) α-fructosylmethionine (j) α-fructosylglutamic acid (k) ε-fructosyllysine (l) β-fructosylalanine. The amadrianse according to (1) or (2) above, which is one or more selected from.
(4) The following (m) to (s): of the amino acid sequence shown in SEQ ID NO: 1.
(M) Arginine at position 62 (n) Leucine at position 63 (o) Arginine at position 64 (p) Tyrosine at position 261 (q) Glycine at position 263 (r) Alanine at position 355 (s) Arginine at position 417 The amadrianase according to (3) above, which has one or more amino acid residue substitutions at positions corresponding to the amino acids selected from the group.
(5) Substitution of the amino acid residue at the position corresponding to one or more amino acids selected from the group consisting of (m) to (s) of the amino acid sequence shown in SEQ ID NO: 1 is from the following (t). (Z):
(T) Arginine at position 62 has been replaced with alanine or serine;
(U) Leucine at position 63 has been replaced with alanine;
(V) Arginine at position 64 has been replaced with serine, aspartic acid, glutamic acid or histidine;
(W) Tyrosine at position 261 has been replaced with phenylalanine, asparagine or lyptophan;
(X) Glycine at position 263 is replaced with serine;
(Y) Alanine at position 355 has been replaced with histidine or aspartic acid;
(Z) Arginine at position 417 has been replaced with histidine;
The amadriase according to (4) above.
(6) In the amino acid sequence shown in SEQ ID NO: 1, the following (aa) to (af):
(Aa) Substitution of tyrosine at position 261 with tryptophan and substitution of glycine at position 263 with serine;
(Ab) Substitution of arginine at position 62 with serine, substitution of tyrosine at position 261 with tryptophan and substitution of glycine at position 263 with serine;
(Ac) Substitution of tyrosine at position 261 with tryptophan; substitution of glycine at position 263 with serine and substitution of alanine at position 355 with histidine;
(Ad) Substitution of tyrosine at position 261 with tryptophan; substitution of glycine at position 263 with serine and substitution of alanine at position 355 with aspartic acid;
(Ae) Substitution of arginine at position 62 with serine, substitution of tyrosine at position 261 with tryptophan, substitution of glycine at position 263 with serine and substitution of alanine at position 355 with histidine;
(Af) Selected from the group consisting of substitution of arginine at position 62 with serine, substitution of tyrosine at position 261 with tryptophan, substitution of glycine at position 263 with serine, and substitution of alanine at position 355 with aspartic acid. The amadriase according to (4) above, which has a substitution of an amino acid residue.
(7) An amadriase gene encoding the amino acid sequence according to any one of (1) to (6) above.
(8) A recombinant vector containing the amadriase gene according to (7) above.
(9) A host cell containing the recombinant vector according to (8) above.
(10) The following steps:
(I) The step of culturing the host cell according to (9) above;
(Ii) A method for producing amadriase, which comprises a step of expressing an amadriase gene contained in a host cell; and (iii) a step of isolating the amadriase from a culture.
(10) A kit for measuring glycated hemoglobin containing the amadriase according to any one of (1) to (6) above.

According to the present invention, an amadriase having excellent substrate specificity, which can be advantageously used as a diagnostic enzyme for diabetes and in a measurement kit for diabetes markers, specifically, "reactivity to αFVH" other than "αFVH". It is possible to provide an amadriase having a reduced proportion of "reactivity to glycated amino acids".

It is a figure which illustrates the sequence of known amadriase derived from various species.

Hereinafter, the present invention will be described in detail.
(Amadriase)
Amadriase is also called ketoamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, or fructosylamine oxidase. In the presence of oxygen, imino2acetic acid or a derivative thereof (amadori compound) is oxidized to glyoxylic acid or α-. An enzyme that catalyzes the reaction that produces ketoaldehyde, amino acids or peptides, and hydrogen peroxide. Amadriase is widely distributed in nature and can be obtained by searching for microorganisms and enzymes of animal or plant origin. In the case of microorganisms, it can be obtained from, for example, filamentous fungi, yeast, bacteria and the like.

In one aspect, the amadriase of the present invention is a variant of the amadriase with modified substrate specificity, made based on the amadriase having the amino acid sequence set forth in SEQ ID NO: 1, 30, 33 or 35. Examples of such variants are SEQ ID NOs: 1, 30, 33 or 35 with high sequence identity (eg, 75% or greater, preferably 80% or greater, more preferably 85% or greater, even more preferably 90%. Above, more preferably 95% or more, still more preferably 97% or more, most preferably 99% or more) in the amadriase and the amino acid sequence of SEQ ID NO: 1, 30, 33 or 35, from 1 to number. Amadriase in which an amino acid has a modified or mutated, or deleted, substituted, added and / or inserted amino acid sequence can be mentioned.

As long as the conditions relating to the amino acid sequence described in the scope of the patent claim are satisfied, for example, the genus Eupenicillium, the genus Pyrenochaeta, the genus Arthrinium, the genus Curvularia, the genus Neocosmospora, the genus Cryptococcus, the genus Phaeosphaeria, the genus Asperulo Penicillium sp., Fusarium sp., Achaetomiella genus, Achaetomium genera, such as Thielavia sp., Chaetomium spp., Gelasinospora genus, Microascus spp., Leptosphaeria species, Ophiobolus genus, Pleospora genus, Coniochaetidium spp., Pichia spp., Corynebacterium spp., Agrobacterium sp., Arthrobacter sp. , It may be prepared based on amadriase derived from other species.
The various sequences shown in FIG. 1 are obtained by arranging several examples of known amino acid sequences of amadriase having 75% or more identity with the sequence of SEQ ID NO: 1 using a known algorithm. The amino acid sequences shown in SEQ ID NO: 1 are shown at the top except for positions 184 and 265. The figure shows the mutation points in the mutant of the present invention.

In FIG. 1, amadriase derived from the genus Coniochaeta, amadriase derived from Eupenicularlium terrenum (SEQ ID NO: 26), Pyrenochaeta sp. Derived ketoamine oxidase (SEQ ID NO: 27), Arthrinium sp. Ketoamine oxidase derived from Ketoamine oxidase (SEQ ID NO: 28), ketoamine oxidase derived from Curvularia clavata (SEQ ID NO: 29), ketoamine oxidase derived from Neocosmospora vasinfecta (SEQ ID NO: 30), fructosyl amino acid oxidase derived from Cryptococcus neoformans (SEQ ID NO: 31). , Fluctosyl peptide oxidase (SEQ ID NO: 32) derived from Phaeosphaeria nodolum, fructosyl amino acid oxidase derived from Aspergillus nidulans, Ulocladium sp. The amino acid sequences of fructosyl amino acid oxidase (SEQ ID NO: 34) derived from and fructosyl amino acid oxidase derived from Penicillium crysogenum are shown.
As can be seen from FIG. 1, the position in the amino acid sequence of the amadriase from another species corresponding to the amino acid at a particular position in the amino acid sequence of the amadriase from the genus Coniochaeta exemplified in SEQ ID NO: 1 is such. A known alignment process based on sequence identity can be easily estimated by those skilled in the art.

In one embodiment, the amadriase modified in reactivity to glycated amino acids other than αFVH is by deleting, inserting, adding, and / or substituting at least one or several amino acid residues in the amino acid sequence of the amadriase. Obtainable.
As a substitution of an amino acid that reduces the reactivity to a saccharified amino acid other than αFVH, we found a substitution of an amino acid at a position corresponding to the amino acid at the following position in the amino acid sequence shown in SEQ ID NO: 1.
(1) Substitution of arginine at position 62, for example, substitution with alanine or serine.
(2) Substitution of leucine at position 63, for example, substitution with alanine.
(3) Substitution of arginine at position 64, for example, substitution with serine, aspartic acid, glutamic acid, histidine.
(4) Substitution of tyrosine at position 261, for example, substitution with phenylalanine, asparagine, tryptophan.
(5) Substitution of glycine at position 263, for example, substitution with serine.
(6) Substitution of alanine at position 355, for example, substitution with histidine or aspartic acid.
(7) Substitution of arginine at position 417, for example, substitution with histidine.

The mutant of amadriase having modified reactivity to a glycated amino acid other than αFVH may have at least one of the above amino acid substitutions or deletions, and may have a plurality of amino acid substitutions or deletions. .. For example, it has 1, 2, 3, 4, 5, 6 or 7 of the above amino acid substitutions.
Among them, mutants having amino acid substitutions corresponding to the following amino acid positions are preferable.
(8) Substitution of tyrosine at position 261 and substitution of glycine at position 263, for example, substitution of tryptophan at position 261 and substitution of glycine at position 263 with serine.
(9) Substitution of arginine at position 62, substitution of tyrosine at position 261 and substitution of glycine at position 263, for example, substitution of arginine at position 62 with serine, substitution of tyrosine at position 261 with tryptophan and glycine at position 263. Replacement with serine.
(10) Substitution of tyrosine at position 261 and substitution of glycine at position 263 and substitution of alanine at position 355, for example, substitution of tyrosine at position 261 with tryptophan, substitution of glycine at position 263 with serine and alanine at position 355. Replacement with histidine.
(11) Substitution of tyrosine at position 261 and substitution of glycine at position 263 and substitution of alanine at position 355, for example, substitution of tyrosine at position 261 with tryptophan, substitution of glycine at position 263 with serine and alanine at position 355. Replacement with aspartic acid.
(12) Substitution of arginine at position 62, substitution of tyrosine at position 261 and substitution of glycine at position 263 and substitution of alanine at position 355, for example, substitution of arginine at position 62 with serine, substitution of tyrosine at position 261 to tryptophan. Substitution of glycine at position 263 with serine and substitution of alanine at position 355 with histidine.
(13) Substitution of arginine at position 62, substitution of tyrosine at position 261 and substitution of glycine at position 263 and substitution of alanine at position 355, for example, substitution of arginine at position 62 with serine, substitution of tyrosine at position 261 to tryptophan. Substitution of glycine at position 263 with serine and alanine at position 355 with aspartic acid.

The amadriase mutant modified in reactivity to a saccharified amino acid other than αFVH of the present invention has an amino acid substitution in the amino acid sequence shown in SEQ ID NO: 1, 30, 33 or 35 that causes the above-mentioned modification of substrate specificity. However, at positions other than those substituted amino acids, one or several more amino acids (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1) are deleted. Containing an inserted, added and / or substituted amino acid sequence, it comprises an amadriase variant having amadriase activity and modified substrate specificity. Further, with respect to the amino acid sequence of the portion of the amino acid sequence shown in SEQ ID NO: 1, 30, 33 or 35 excluding amino acids other than the substituted amino acid, which has an amino acid substitution variation resulting in the above-mentioned modification of substrate specificity. , 75% or more, preferably 86% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 97% or more, most preferably 99% or more amino acid sequences having amino acid sequence identity. Includes amadriase variants with amadriase activity and modified substrate specificity.

In the above amino acid substitution, the amino acid position is substituted in the amino acid sequence of Amadriase derived from the genus Coniochaeta shown in SEQ ID NO: 1 at the position corresponding to the position in the amino acid sequence shown in SEQ ID NO: 1. The meaning of "corresponding position" will be described later. The amadriase having the amino acid sequence shown in SEQ ID NO: 1 is the genus Coniochaeta produced by Escherichia coli carrying a recombinant plasmid (deposit number: FERM BP-10593) referred to as pKK223-3-CFP-T7 in the present specification. Derived from the plasmid (CFP-T7), which is a modified plasmid with excellent thermal stability previously found by the applicant (see International Patent No. 2007/125779). CFP-T7 is a triple mutant obtained by sequentially introducing artificial mutations at positions 272, 302 and 388 with respect to the native Amadriase derived from the genus Coniochaeta.

(Amino acid sequence identity)
Amino acid sequence identity can be calculated using any known method. For example, GENETYX Ver. It can be calculated by a program such as maximum matching or search homology of 11 (manufactured by Genetics) or a program such as maximum matching or multiple alignment of DNASIS Pro (manufactured by Hitachi Software).

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

Next, a method of synthesizing an appropriate probe DNA based on the amino acid sequence of the amadriase and selecting the amadriase gene from a library of chromosomal DNA or cDNA using this, or an appropriate primer DNA based on the amino acid sequence. The DNA containing the gene fragment of interest encoding the amadriase is amplified by an appropriate polymerase chain reaction (Polymerase Chain Reaction, PCR method) such as the 5'RACE method or the 3'RACE method. Can be ligated to obtain DNA containing the full length of the target amadriase gene.
As a preferable example of the gene encoding the amadriase thus obtained, there is an amadriase gene derived from the genus Coniocaeta (see JP-A-2003-235585).

It is preferable in terms of handling that these amadriase genes are linked to various vectors as usual. For example, Coniochaeta sp. Examples thereof include the recombinant plasmid pKK223-3-CFP (see JP-A-2003-235585) in which the DNA encoding the amadriase gene derived from the NISL 9330 strain is inserted into pKK223-3 Vector (manufactured by Amersham Biotech).

(vector)
The vector that can be used in the present invention is not limited to the above-mentioned plasmid, and any other vector known to those skilled in the art such as bacteriophage and cosmid can be used. Specifically, for example, pBluescriptII SK + (manufactured by Stratagene) and the like are preferable.

(Mutation treatment of amadriase gene)
The mutation treatment of the amadriase gene can be carried out by any known method depending on the intended mutation form. That is, a wide range of methods such as a method of contacting and acting the amadriase gene or a recombinant DNA in which the gene is integrated with a drug that is a mutagen; an ultraviolet irradiation method; a genetic engineering method; or a method that makes full use of a protein engineering method, etc. Can be used.

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

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

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

(Transformation / transduction)
The amadriase gene obtained as described above is incorporated into a vector such as a bacteriophage, 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 commonly used. Can be transformed or transduced. For example, as a host, a microorganism belonging to the genus Escherichia, for example, the obtained recombinant DNA is used to transduce, for example, Escherichia coli K-12 strain, preferably Escherichia coli JM109 strain, Escherichia coli DH5α strain (both manufactured by Takara Bio), and the like. Each strain is obtained by conversion or transduction into them.

(Identification of the position corresponding to the amino acid)
The "position corresponding to an amino acid" refers to a position in the amino acid sequence of an amadriase derived from another species corresponding to an amino acid at a specific position in the amino acid sequence of the amadriase derived from the genus Coniocaeta shown in SEQ ID NO: 1.
As a method for identifying the "position corresponding to an amino acid", the amino acid sequences are compared using a known algorithm such as the Lippmann-Person method, and the maximum identity is the same as the conserved amino acid residue present in the amino acid sequence of each amadriase. It can be done by giving sex. By aligning the amino acid sequence of amadriase in this way, it is possible to determine the position of homologous amino acid residues in each amadriase sequence in the sequence regardless of insertion or deletion in the amino acid sequence. Homological positions are considered to be co-located in the three-dimensional structure and can be presumed to have similar effects on the specific function of the amadriase of interest.

In the present invention, the "position corresponding to arginine at position 62 of the amino acid sequence shown in SEQ ID NO: 1" refers to the determined amino acid sequence of amadriase as the amino acid sequence of amadriase derived from the genus Coniocaeta shown in SEQ ID NO: 1. When compared, it means the amino acid corresponding to the arginine at position 62 of the amadriase of SEQ ID NO: 1. Thereby, the amino acid sequence can be aligned and specified by the above-mentioned method for specifying the "amino acid at the corresponding position".
That is, Pyrenochaeta sp., An amadriase derived from Eupenicularlium terrenum. Derived ketoamine oxidase, Arthrinium sp. Ketoamine oxidase from Cryptococcus neoformans, Fructosyl amino acid oxidase from Cryptococcus neoformans, Ulocladium sp., Ketoamine oxidase from Curvalaria clavata, ketoamine oxidase from Neocosmospora vasinfecta. Fructosyl amino acid oxidase derived from Pelicillium crysogenum, arginine at position 62, fructosyl peptide oxidase derived from Phaeosphaeria nodolum, serine at position 62, and arginine derived from Aspergillus nidulans, arginine at position 61. ..

Further, in the present invention, the "position corresponding to leucine at position 63 of the amino acid sequence shown in SEQ ID NO: 1" refers to the determined amino acid sequence of amadriase as the amino acid sequence of amadriase derived from the genus Coniocaeta shown in SEQ ID NO: 1. When compared, it means the amino acid corresponding to leucine at position 63 of the amadriase of SEQ ID NO: 1. Thereby, the amino acid sequence can be aligned and specified by the above-mentioned method for specifying the "amino acid at the corresponding position".
That is, Pyrenochaeta sp., An amadriase derived from Eupenicularlium terrenum. Derived ketoamine oxidase, Arthrinium sp. Ketoamine oxidase derived from Ketoamine oxidase, ketoamine oxidase derived from Curvularia clavata, ketoamine oxidase derived from Neocosmospora vasinfecta, fructosyl peptide oxidase derived from Phaeosphaeria nodolum, Ulocladium sp. Fructosyl amino acid oxidase derived from Pencillium crysogenum is leucine at position 63, fructosyl amino acid oxidase derived from Cryptococcus neoformans is isoleucine at position 63, and fructosyl amino acid oxidase derived from Aspergillus nidulans is at position 62. ..

Further, "the position corresponding to arginine at position 64 of the amino acid sequence shown in SEQ ID NO: 1" is when the determined amino acid sequence of amadriase is compared with the amino acid sequence of amadriase derived from the genus Coniocaeta shown in SEQ ID NO: 1. , Means the amino acid corresponding to the arginine at position 64 of the amadriase of SEQ ID NO: 1. This can also be specified by aligning the amino acid sequences by the above method.
That is, Pyrenochaeta sp., An amadriase derived from Eupenicularlium terrenum. Derived ketoamine oxidase, Arthrinium sp. Ketoamine oxidase derived from Ketoamine oxidase, ketoamine oxidase derived from Curvularia clavata, ketoamine oxidase derived from Neocosmospora vasinfecta, fructosyl peptide oxidase derived from Phaeosphaeria nodolum, Ulocladium sp. The fructosyl amino acid oxidase derived from Penicillium crysogenum, the fructosyl amino acid oxidase derived from Cryptococcus neoformans is arginine at position 64, and the fructosyl amino acid oxidase derived from Aspergillus nidulans is arginine at position 63.

Further, "the position corresponding to tyrosine at position 261 of the amino acid sequence shown in SEQ ID NO: 1" is when the confirmed amino acid sequence of amadriase is compared with the amino acid sequence of amadriase derived from the genus Coniocaeta shown in SEQ ID NO: 1. , Means the amino acid corresponding to the tyrosine at position 261 of the amadriase of SEQ ID NO: 1. This can also be specified by aligning the amino acid sequences by the above method.
That is, Arthrinium sp., An amadriase derived from Eupenicularium terrenum. Ketoamine oxidase derived from ketoamine oxidase, ketoamine oxidase derived from Neocosmospora vasinfecta, fructosyl amino acid oxidase derived from Cryptococcus neoformans, fructosyl amino acid oxidase derived from Aspergillus nidulans, fructosyl amino acid oxidase derived from Aspergillus nidulans, fluctosyl amino acid oxidase derived from Penicillium crysogenum Ketoamine oxidase from Curvularia clavata, ketoamine oxidase from Curvularia sp. The derived fructosyl amino acid oxidase is tyrosine at position 259, and the fructosyl peptide oxidase derived from Phaeosphaeria nodom is tyrosine at position 257.

Further, "the position corresponding to glycine at position 263 of the amino acid sequence shown in SEQ ID NO: 1" is when the confirmed amino acid sequence of amadriase is compared with the amino acid sequence of amadriase derived from the genus Coniocaeta shown in SEQ ID NO: 1. , Means the amino acid corresponding to the glycine at position 263 of the amadriase of SEQ ID NO: 1. Thereby, the amino acid sequence can be aligned and specified by the above-mentioned method for specifying the "amino acid at the corresponding position".
That is, Arthrinium sp., An amadriase derived from Eupenicularium terrenum. Ketoamine oxidase derived from ketoamine oxidase, ketoamine oxidase derived from Neocosmospora vasinfecta, fructosyl amino acid oxidase derived from Aspergillus nidulans, glycine at position 263 in Fluxtosyl amino acid oxidase derived from Penicillium crysogenum, Pyrenochaeta sp. Ketoamine oxidase from Curvularia clavata, ketoamine oxidase from Curvularia sp. The derived fructosyl amino acid oxidase is glycine at position 261, the fructosyl amino acid oxidase derived from Cryptococcus neoformans is serine at position 263, and the fructosyl peptide oxidase derived from Phaeosphaeria nodom is glycine at position 259.

Further, "the position corresponding to alanine at position 355 of the amino acid sequence shown in SEQ ID NO: 1" is when the determined amino acid sequence of amadriase is compared with the amino acid sequence of amadriase derived from the genus Coniocaeta shown in SEQ ID NO: 1. , Means the amino acid corresponding to alanine at position 355 of the amadriase of SEQ ID NO: 1. Thereby, the amino acid sequence can be aligned and specified by the above-mentioned method for specifying the "amino acid at the corresponding position".
That is, amadriase derived from Eupenicillium terrenum, fructosyl amino acid oxidase derived from Aspergillus nidulans, fructosyl amino acid oxidase derived from Cryptococcus neoformans, fructosyl amino acid oxidase derived from Penicillium crysogenum, and fructosyl amino acid oxidase derived from Penicillium crysogenum at position 355. Ketoamine oxidase from Curvularia clavata, ketoamine oxidase from Curvularia sp. In the derived fructosyl amino acid oxidase, alanine at position 353, Arthrinium sp. The derived ketoamine oxidase is alanine at position 356, the ketoamine oxidase derived from Neocosmospora vasinfecta is serine at position 355, and the fructosyl peptide oxidase derived from Phaeosphaeria nodolum is alanine at position 351.

Further, the "position corresponding to arginine at position 417 of the amino acid sequence shown in SEQ ID NO: 1" is when the determined amino acid sequence of amadriase is compared with the amino acid sequence of amadriase derived from the genus Coniocaeta shown in SEQ ID NO: 1. , Means the amino acid corresponding to arginine at position 417 of the amadriase of SEQ ID NO: 1. Thereby, the amino acid sequence can be aligned and specified by the above-mentioned method for specifying the "amino acid residue at the corresponding position".

That is, arginine at position 417 in the amadriase derived from Eupenicularlium terrenum, Pyrenochaeta sp. Ketoamine oxidase from Curvularia clavata, ketoamine oxidase from Curvularia sp. In the derived fructosyl amino acid oxidase, arginine at position 416, Arthrinium sp. Arginine at position 419 for ketoamine oxidase derived from ketoamine oxidase, isoleucine at position 418 for ketoamine oxidase derived from Neocosmospora vasinfecta, fructosyl amino acid oxidase derived from Cryptococcus neoformans, fructosyl amino acid oxidase derived from Aspergillus nidulanes and fructosyl amino acid oxidase derived from It is arginine at position 414 in fructosyl peptide oxidase and lysine at position 417 in fructosyl amino acid oxidase derived from Pencillium crysogenum.

(Production of Amadriase of the present invention)
In order to produce the amadriase using the strain having the ability to produce the amadriase with the improved substrate specificity obtained as described above, this strain may be cultured by a usual solid culture method. It is preferable to use the liquid culture method for culturing as much as possible.

As a medium for culturing the above strains, for example, sodium chloride and dihydrogen phosphate are added to one or more nitrogen sources such as yeast extract, tripton, peptone, meat extract, corn steep liquor, or soybean or wheat bran leachate. Add one or more of inorganic salts such as potassium, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate, and if necessary, add sugar raw materials, vitamins, etc. as appropriate. Is used.

In addition, the amount of the target enzyme produced by adding a substrate on which the amadriase can act or a similar compound thereof, for example, glycated amino acids, glycated peptides, glycated protein degradation products, or glycated proteins such as glycated hemoglobin and glycated albumin to the medium. Can be improved.

It is appropriate to adjust the initial pH of the medium to pH 7-9. The culture is carried out at a culture temperature of 20 to 42 ° C., preferably a culture temperature of about 37 ° C. for 4 to 24 hours, more preferably a culture temperature of about 37 ° C. for 8 to 16 hours, aeration-stirred deep culture, shaking culture, and standing. It is preferable to carry out by culturing or the like.

After completion of the culture, in order to collect amadriase from the culture, it can be obtained by using a usual enzyme collection means. For example, the cells are subjected to ultrasonic destruction treatment, grinding treatment, etc. by a conventional method, or this enzyme is extracted using a lytic enzyme such as lysozyme, or the bacterium is shaken or left to stand in the presence of toluene or the like. This enzyme can be excreted from the cells. Then, the solid portion is removed by filtering, centrifuging, etc. of this solution, and if necessary, the nucleic acid is removed with streptomycin sulfate, protamine sulfate, manganese sulfate, etc., and then ammonium sulfate, alcohol, acetone, etc. are added thereto. Fractionate, and the precipitate is collected to obtain the crude enzyme of amadriase.

In order to further obtain an amadriase purified enzyme preparation from the above crude enzyme of amadriase, for example, a gel filtration method using Sephadex, Superdex or Ultrogel, an ion exchange carrier, a hydrophobic carrier, an adsorption elution method using hydroxyapatite, etc. An electrophoresis method using a polyacrylamide gel or the like, a precipitation method such as a sugar density gradient centrifugation method, an affinity chromatography method, a fractionation method using a molecular sieving membrane or a hollow yarn membrane, etc. are appropriately selected, or a combination thereof is carried out. By doing so, a purified amadriase enzyme preparation can be obtained. In this way, amadriase with reduced reactivity to the desired glycated amino acid can be obtained.

(Reduction of reactivity with glycated amino acids other than αFVH in the amadriase of the present invention)
As a result of mutation in the amino acid sequence of the amadriase of the present invention obtained by the above means, the reactivity to saccharified amino acids other than αFVH is lowered as compared with the one before the modification. As a result, the substrate specificity for αFVH is improved. Specifically, the ratio of "reactivity to various glycated amino acids other than αFVH" to "reactivity to αFVH", for example, the ratio of "reactivity to αFL" is reduced as compared with the one before modification. It is characterized by being.
The "glycated amino acid" is not particularly limited as long as it is an amino acid or a short-chain peptide that has been glycated, but for example, various α-glycated amino acids that are assumed to be present in a free state in blood or infusion. , Β-glycated amino acids and / or ε-glycated amino acids.
Specifically, for example, α-fructosyl leucine (αFL), α-fructosylalanine (αFA), α-fructosylglycine (αFG), α-fructosylvaline (αFV), α-fructosyl isoleucine (αFV). αFI), α-fructosyl leucine (αFT), α-fructosylphenylalanine (αFF), α-fructosylserine (αFS), α-fructosylmethionine (αFM), α-fructosylglutamic acid (αFE), α- Fructosyl lysine, α-fructosyl tryptophan, α-fructosyl cysteine, α-fructosyl tyrosine, α-fructosyl arginine, α-fructosyl histidine, α-fructosylproline, α-fructosyl aspartic acid, ε-fluc Examples thereof include tosyllysine (εFK), α, ε-fructosyllysine and β-fructosylalanine (βFA).
These glycated amino acids can be brought into the blood, for example, in a free state by infusion or drug administration, or can be produced by the reaction of sugar and amino acids in the blood. Alternatively, in the pretreatment for measuring glycated hemoglobin, it can be cut out as a glycated amino acid by the action of protease, peptidase, etc. from a glycated protein such as glycated hemoglobin or glycated albumin.
For example, the infusion solution contains a large amount of αFL, αFA, αFG, αFV, etc., and these are assumed to be typical examples of various glycated amino acids other than αFVH when measuring a sample of a patient receiving the infusion solution. obtain.
In the present invention, the glycated amino acid may include a short chain glycated peptide that may cause a measurement error in the method of measuring HbA1c by measuring αFVH.

In the decrease in reactivity with glycated amino acids other than αFVH in the amadriase of the present invention, it is particularly preferable that the reactivity with αFL, which is an error-causing substance that can be produced from L-leucine and glucose, is reduced. αFL is most abundant in high calorie amino acid infusions.
Specifically, in the amadriase of the present invention, "αFL / αFVH" indicating the ratio of the reactivity to αFL to the reactivity to αFVH is 13% or more, preferably 23% or more, more preferably than before the modification. Is preferably reduced by 38% or more, more preferably 72% or more, and most preferably 83% or more. That is, when the ratio before modification is 100, the reactivity ratio after the introduction of the mutation is reduced to 87% or less, preferably 77% or less, more preferably 62% or less, and further. It is preferably reduced to 28% or less, and most preferably 17% or less.

It is also preferable that the reactivity of the amadriase of the present invention with respect to glycated amino acids other than αFVH is reduced with respect to αFA, which is an error-causing substance that can be produced from L-alanine and glucose. αFA is abundant in high-calorie amino acid infusions.
Specifically, in the amadriase of the present invention, "αFA / αFVH" indicating the ratio of reactivity to αFA to reactivity to αFVH is 6% or more, preferably 33% or more, more preferably than before modification. Is preferably reduced by 46% or more, more preferably 53% or more, further preferably 67% or more, and most preferably 72% or more. That is, when the ratio before modification is 100, the reactivity ratio after the introduction of the mutation is reduced to 94% or less, preferably 67% or less, more preferably 54% or less, and further. It is preferably reduced to 47% or less, more preferably 33% or less, and most preferably 28% or less.

Furthermore, it is also preferable as a property of the amadriase of the present invention that the reactivity with αFG, which is an error-causing substance that can be produced from glycine and glucose, which are abundantly present in high-calorie amino acid infusions, is reduced.
Specifically, in the amadriase of the present invention, αFG / αFVH showing the ratio of reactivity to αFG to reactivity to αFVH is 19% or more, preferably 31% or more, more preferably 50, as compared with that before modification. % Or more, more preferably 69% or more, and most preferably 75% or more. That is, when the ratio before modification is 100, the reactivity ratio after the introduction of the mutation is reduced to 81% or less, preferably 69% or less, more preferably 50% or less, and further. It is preferably reduced to 31% or less, and most preferably to 25% or less.

Furthermore, it is also preferable as a property of the amadriase of the present invention that the reactivity with αFV, which is an error-causing substance that can be produced from L-valine and glucose, which are abundantly present in high-calorie amino acid infusions, is reduced.
Specifically, in the amadriase of the present invention, αFV / αFVH showing the ratio of reactivity to αFV to reactivity to αFVH is 6% or more, preferably 19% or more, more preferably 37 than before modification. % Or more, more preferably 49% or more, and most preferably 56% or more. That is, when the ratio before modification is 100, the reactivity ratio after the introduction of the mutation is reduced to 94%, preferably to 81%, more preferably to 63%, and further preferably 51. It is preferably reduced to%, most preferably to 44%.

The ratio of reactivity to saccharified amino acid (for example, αFL), which is a substance causing measurement error as described above, to reactivity to αFVH, which is a true measurement target substance, can be determined under arbitrary conditions using a known measurement method for amadriase. It can be measured below and compared to the one before modification. For example, at pH 7.0, the activity measured by adding 5 mM αFL is divided by the activity measured by adding 5 mM αFVH to determine the ratio of reactivity to αFL to reactivity to αFVH. It can be calculated and compared between the one before modification and the one after modification.

As an example of the amadriase of the present invention having improved substrate specificity as compared with the one before modification, for example, it is produced by Escherichia coli JM109 (pKK223-3-CFP-T7-62S / 261W / 263S / 355D) strain. Amadriase is mentioned. Amadriase having such improved substrate specificity is a glycated amino acid derived from the β-chain amino terminus of HbA1c, with a good reduction in the degree to which glycated amino acids such as αFL, αFA, αFG and / or αFV are measured as noise. Since it is possible to measure only a certain αFVH, it is possible to perform highly accurate measurement, which is very advantageous for industrial use.

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

Examples of the method for measuring the enzymatic activity of amadriase in the present invention include a method for measuring the amount of hydrogen peroxide produced by the enzymatic reaction and a method for measuring the amount of oxygen consumed by the enzymatic reaction. The method for measuring the amount of hydrogen peroxide is shown below as an example.

Unless otherwise specified, αFL, αFA, αFG, αFV, or αFVH is used as a substrate for measuring the activity of amadriase in the present invention. The enzyme titer is defined as 1 U as the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute when measured using αFL, αFA, αFG, αFV, or αFVH as a substrate.
As the saccharified amino acid such as αFL and the saccharified peptide such as αFVH, those synthesized and purified based on the methods of Sakagami et al., Hashiba and Heins et al. Can be used (Japanese Patent Laid-Open No. 2001-95598, Agric. Biol. See Chem., 42, 763-768 (1978), Carbohydrate Research, 116, 183-195 (1983)).

A. : Reagent preparation Reagent 1: Peroxidase, 4-aminoantipyrine solution 5.0 kU of peroxidase (manufactured by Kikkoman Biochemifa), 100 mg of 4-aminoantipyrine (manufactured by Tokyo Kasei) at 0.1 M potassium phosphate buffer Dissolve in a solution (pH 7.5, pH 7.0 or pH 6.5) and formulate to 1,000 ml.
Reagent 2: TOOS solution 500 mg of TOOS (manufactured by Dojin Chemical Laboratory Co., Ltd.) is dissolved in ion-exchanged water and the volume is adjusted to 100 ml.
Reagent 3: Substrate solution (150 mM; final concentration 5 mM)
625 mg of αFVH, 440 mg of αFL, 377 mg of αFA, 355 mg of αFG, or 419 mg of αFV are dissolved in ion-exchanged water and the volume is adjusted to 10 ml.

B: Activity measurement method 2.7 ml of reagent 1, 100 μl of reagent 2, and 100 μl of enzyme solution are mixed and preheated at 37 ° C. for 5 minutes. Then, 100 μl of the reagent 3 was added and mixed well, and then the change with time of the absorbance at 555 nm was observed with a spectrophotometer (U-3010A, manufactured by Hitachi High-Technologies Corporation), and the amount of change in the absorbance at 555 nm per minute ( ΔAs) was measured. In the control solution, the amount of change (ΔA0) in absorbance at 555 nm per minute 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 active 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 of the reaction solution per minute ΔA0: Change in absorbance of the control solution per minute
39.2: mmol extinction coefficient of quinoneimine dye produced by the reaction (mM ^-1 · cm ^-1 )
Number of moles of quinoneimine dye produced by 0.5: 1 mol of hydrogen peroxide
df: Dilution coefficient

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the technical scope of the present invention is not limited to those examples.

[Example 1]
(Preparation of amadriase derived from the genus Coniochaeta)
(1) Preparation of recombinant plasmid pKK223-3-CFP-T7 DNA Escherichia coli JM109 (pKK223-3-CFP-T7) strain having a recombinant plasmid of the amadriase gene (SEQ ID NO: 2) derived from the genus Coniochaeta (International Publication No. 2) (See 2007/125779) in 3 ml of LB-amp medium [1% (w / v) plasmid, 0.5% (w / v) peptone, 0.5% (w / v) NaCl, 50 μg / Inoculated into ml ampicillin] and cultured with shaking at 37 ° C. for 16 hours to obtain a culture.
This culture was centrifuged at 10,000 × g for 1 minute to collect cells to obtain bacterial cells. Recombinant plasmid pKK223-3-CFP-T7 was extracted and purified from this cell using GenElute Plasmid Mini-Prep Kit (manufactured by Sigma-Aldrich), and 2.5 μg of recombinant plasmid pKK223-3-3 was extracted. CFP-T7 DNA was obtained.

(2) Site-specific modification operation of recombinant plasmid pKK223-3-CFP-T7 DNA Using the obtained recombinant plasmid pKK223-3-CFP-T7 DNA as a template, synthetic oligonucleotides of SEQ ID NOs: 3 and 4, A PCR reaction was carried out using KOD-Plus- (manufactured by Toyo Spinning Co., Ltd.) under the following conditions.
That is, 5 μl of 10 × KOD-Plus-buffer solution, 5 μl of dNTPs mixed solution prepared to have dNTPs of 2 mM each, 2 μl of 25 mM sulfonyl ₄ solution, and 50 ng of pKK223-3-CFP-T7 DNA as a template. , 15 pmol of each of the above synthetic oligonucleotides and 1 unit of KOD-Plus- were added, and the total amount was adjusted to 50 μl with sterilized water. The prepared reaction solution was incubated at 94 ° C. for 2 minutes using a thermal cycler (manufactured by Eppendorf), followed by "94 ° C., 15 seconds"-"50 ° C., 30 seconds"-"68 ° C., 6 minutes". The cycle of 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 BIOLABBS), the remaining template DNA was cleaved, and 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 the DNA encoding amadrianase 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 position 62 of the amino acid sequence shown in SEQ ID NO: 1 was determined. A recombinant plasmid (pKK223-3-CFP-T7-62A) encoding a modified amadrianase in which arginine was replaced with alanine was obtained.

Subsequently, in order to replace the arginine at position 62 of the amino acid sequence shown in SEQ ID NO: 1 with serine, the synthetic oligonucleotide of SEQ ID NO: 4 and 5, KOD, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-62S) encoding a modified amadriase in which arginine at position 62 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with serine was obtained.

Subsequently, in order to replace leucine at position 63 of the amino acid sequence shown in SEQ ID NO: 1 with alanine, the synthetic oligonucleotide of SEQ ID NO: 6 and 7, KOD, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-63A) encoding a modified amadrianase in which leucine at position 63 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with alanine was obtained.

Subsequently, in order to replace the arginine at position 64 of the amino acid sequence shown in SEQ ID NO: 1 with serine, the synthetic oligonucleotide of SEQ ID NOs: 8 and 9, KOD, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-64S) encoding a modified amadriase in which arginine at position 64 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with serine was obtained.

Subsequently, in order to replace the arginine at position 64 of the amino acid sequence shown in SEQ ID NO: 1 with aspartic acid, the synthetic oligonucleotides of SEQ ID NOs: 9 and 10 were prepared using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using KOD-Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-64D) encoding a modified amadriase in which arginine at position 64 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with aspartic acid was obtained.

Subsequently, in order to replace the arginine at position 64 of the amino acid sequence shown in SEQ ID NO: 1 with glutamic acid, the synthetic oligonucleotide of SEQ ID NOs: 9 and 11, KOD, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-64E) encoding a modified amadriase in which arginine at position 64 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with glutamic acid was obtained.

Subsequently, in order to replace the arginine at position 64 of the amino acid sequence shown in SEQ ID NO: 1 with histidine, the synthetic oligonucleotides of SEQ ID NOs: 9 and 12, KOD, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-64H) encoding a modified amadriase in which arginine at position 64 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with histidine was obtained.

Subsequently, in order to replace the tyrosine at position 261 of the amino acid sequence shown in SEQ ID NO: 1 with phenylalanine, the synthetic oligonucleotides of SEQ ID NOs: 13 and 14, KOD, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-261F) encoding a modified amadriase in which the tyrosine at position 261 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with phenylalanine was obtained.

Subsequently, in order to replace the tyrosine at position 261 of the amino acid sequence shown in SEQ ID NO: 1 with asparagine, the synthetic oligonucleotides of SEQ ID NOs: 14 and 15, KOD, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-261N) encoding a modified amadriase in which the tyrosine at position 261 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with asparagine was obtained.

Subsequently, in order to replace the tyrosine at position 261 of the amino acid sequence shown in SEQ ID NO: 1 with tryptophan, the synthetic oligonucleotides of SEQ ID NOs: 14 and 16 and KOD were used as a template using the recombinant plasmid pKK223-3-CFP-T7 DNA. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-261W) encoding a modified amadriase in which the tyrosine at position 261 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with tryptophan was obtained.

Subsequently, in order to replace the glycine at position 263 of the amino acid sequence shown in SEQ ID NO: 1 with serine, the synthetic oligonucleotides of SEQ ID NOs: 17 and 18, KOD, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-263S) encoding a modified amadriase in which glycine at position 263 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with serine was obtained.

Subsequently, in order to replace the alanine at position 355 of the amino acid sequence shown in SEQ ID NO: 1 with histidine, the synthetic oligonucleotide of SEQ ID NOs: 19 and 20, KOD, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-355H) encoding a modified amadriase in which alanine at position 355 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with histidine was obtained.

Subsequently, in order to replace the alanine at position 355 of the amino acid sequence shown in SEQ ID NO: 1 with aspartic acid, the synthetic oligonucleotides of SEQ ID NOs: 20 and 21 were prepared using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using KOD-Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-355D) encoding a modified amadriase in which alanine at position 355 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with aspartic acid was obtained.

Subsequently, in order to replace arginine at position 417 of the amino acid sequence shown in SEQ ID NO: 1 with histidine, the synthetic oligonucleotides of SEQ ID NOs: 22 and 23, KOD, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template. Using -Plus- (manufactured by Toyo Spinning Co., Ltd.), PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed under the same conditions as above. As a result, a recombinant plasmid (pKK223-3-CFP-T7-417H) encoding a modified amadriase in which arginine at position 417 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with histidine was obtained.

(3) Production of various modified amadriase Escherichia coli JM109 strains carrying the above recombinant plasmids obtained by the above procedure were added to 3 ml of LB-amp medium supplemented with 0.1 mM IPTG at 30 ° C. 16 Cultured for hours. Each of the obtained cultured cells was washed with 20 mM potassium phosphate buffer (pH 7.0), suspended in the buffer, ultrasonically crushed, and centrifuged at 20,000 × g for 10 minutes. Then, 0.6 ml of an enzyme solution for confirming the substrate specificity was prepared.

(4) Measurement of αFL / αFVH, αFA / αFVH, αFG / αFVH and αFV / αFVH Using the above-mentioned enzyme solution, αFL, αFA, αFG or αFV with respect to αFVH by the method shown in the above B: activity measurement method. The enzyme activity of was measured. For comparison, the same measurement was also performed on the unmodified amadriase produced from the Escherichia coli JM109 (pKK223-3-CFP-T7) strain. For the activity measurement, a reagent 1: peroxidase and 4-aminoantipyrine solution adjusted to pH 7.0 were used.
As a result, the αFL / αFVH of the unmodified amadriase produced by the Escherichia coli JM109 (pKK223-3-CFP-T7) strain obtained from the above-mentioned enzyme activity measurement results was 1.53, and the αFA / αFVH was 2. It was .86, αFG / αFVH was 0.16, and αFV / αFVH was 3.07.
On the other hand, αFL / αFVH of various modified amadriase prepared by site-specific mutagenesis, and αFL / αFVH of modified amadriase when the αFL / αFVH value of the amadriase before modification was set to 100%. The ratio was calculated. Similarly, calculations were made for αFA / αFVH, αFG / αFVH, and αFV / αFVH, and the results are shown in Table 1. In the data in the table, the upper value is the ratio of the reactivity of various glycated amino acids to the reactivity of αFVH, and the lower value is the reactivity after the introduction of mutation when the reactivity of CFP-T7 is 100. It is a ratio.

That is, as shown in Table 1, under the conditions of this example, when αFL / αFVH of CFP-T7 is set to 100%, αFL / αFVH of all modified amadriase except CFP-T7-64H is All of them decreased to 87% or less, most notably decreased to 77%, particularly in CFP-T7-62S and CFP-T7-64D, decreased to 62%, and further decreased in CFP-T7-261F. Was reduced to 28%, and further decreased to 17% in CFP-T7-261W, indicating that the substrate specificity was improved.
When αFL / αFVH of modified amadriase decreases to 65% or less when αFL / αFVH of CFP-T7 is set to 100%, αFVH reacts preferentially over αFL which can be contained most in the infusion solution. Therefore, such modifications are considered to be particularly useful.
Similarly, when αFA / αFVH of CFP-T7 was set to 100%, αFA / αFVH of all modified amadriase except CFP-T7-62A decreased to 94% or less, which is remarkable. In particular, it decreased to 67% in CFP-T7-355H, further decreased to 47% in CFP-T7-62S, and further decreased to 33% in CFP-T7-355D. It decreased, and further decreased to 28% in CFP-T7-261W, indicating that the substrate specificity was improved.
Similarly, when the αFG / αFVH of CFP-T7 was set to 100%, the αFG / αFVH of all the modified amadriase shown in Table 1 decreased to 81% or less, and more notably. It decreased to 69% or less, especially in CFP-T7-355D, decreased to 50%, further decreased to 31% in CFP-T7-64E and CFP-T7-261F, and further decreased to CFP-T7-62S. , CFP-T7-64S, CFP-T7-64D and CFP-T7-261W decreased to 25%, indicating that the substrate specificity was improved.
Furthermore, similarly, when the αFV / αFVH of CFP-T7 was set to 100%, the αFV / αFVH of all the modified amadriase shown in Table 1 decreased to 94% or less, which was not remarkable. It decreased to 81%, especially in CFP-T7-62S, decreased to 63%, further decreased to 51% in CFP-T7-261W and CFP-T7-355H, and further decreased in CFP-T7-261F. Was reduced to 44%, indicating that substrate specificity was improved.
Furthermore, CFP-T7-355D was reduced to at least 69% or less in all of αFL / αFVH, αFA / αFVH, αFG / αFVH and αFV / αFVH as compared with CFP-T7, and similarly CFP-T7-62S. Was reduced to at least 63%, and CFP-T7-261W was also a useful mutation reduced to at least 51%.
On the other hand, CFP-T7-64H was found to have improved reactivity with αFL, although the reaction to αFA, αFG and αFV was significantly reduced as compared with CFP-T7. Further, it was found that the reactivity of CFP-T7-62A to αFA was improved even though the reaction to αFL, αFG and αFV was remarkably reduced as compared with CFP-T7.
In this way, by introducing substitutions at several positions, it was possible to reduce the reaction to αFG and αFV, which can be error-causing substances when measuring αFVH. In addition, although there were differences in the presence or absence and degree of effect depending on the position of substitution and the type of amino acid, we found multiple substitutions that can reduce the reactivity to αFL or αFA.

In addition, in International Publication No. 2004/104203, by substituting arginine at position 62 of ketoamine oxidase derived from carbraria clavata YH923 with histidine, εFZK (ε-fructosyl- (α-benzyloxy)) with respect to reactivity to αFVH was used. Since it was disclosed that εFZK / αFVH, which is the ratio of reactivity to carbonyllysine), is reduced from 0.95 to 0.24, when arginine at position 62 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with histidine. The effect of was also verified.
Specifically, using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template and using the synthetic oligonucleotide of SEQ ID NOs: 4 and 24, KOD-Plus- (manufactured by Toyo Spinning Co., Ltd.), under the same conditions as above. PCR reaction, transformation of Escherichia coli JM109, and sequencing of the DNA encoding amadriase in the plasmid DNA retained by the growing colonies were performed. As a result, a recombinant plasmid (pKK223-3-CFP-T7-62H) encoding a modified amadriase in which arginine at position 62 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with histidine was obtained. The obtained recombinant plasmid was cultured by the above method to prepare 0.6 ml of a crude enzyme solution of various modified amadriase.

C. Preparation of Reagent Reagent 4: Substrate solution (60 mM; final concentration 2 mM)
Dissolve 250 mg of αFVH and 185 mg of εFK in ion-exchanged water and adjust the volume to 10 ml.

Using the above-mentioned enzyme solution, the enzyme activity of FK with respect to αFVH was measured by the method using reagent 4 instead of reagent 3 in the above-mentioned B: activity measurement method. For comparison, the same measurement was also performed on the unmodified amadriase produced from the Escherichia coli JM109 (pKK223-3-CFP-T7) strain. For the activity measurement, a reagent 1: peroxidase and 4-aminoantipyrine solution adjusted to pH 7.5 were used.
As a result, the εFK / αFVH of the unmodified amadriase produced by the Escherichia coli JM109 (pKK223-3-CFP-T7) strain was 0.18, and was produced by the Escherichia coli JM109 (pKK223-3-CFP-T7-62H) strain. The εFK / αFVH of amadriase after the introduction of the mutation in which the amino acid at position 62 was replaced with histidine was 0.26. That is, in CFP-T7, substituting arginine at position 62 with histidine adversely deteriorates substrate specificity, which is contrary to the finding of substrate specificity modification using εFZK in ketoamine oxidase derived from carbraria clavata YH923. It was confirmed that the effect of

[Example 2]
(Accumulation of mutations effective for improving substrate specificity)
Using the various recombinant plasmid DNAs shown in Table 2 as templates, synthetic oligonucleotides (SEQ ID NOs: 4, 5, 18, 19, 20, 21, 25) and KOD-Plus- (manufactured by Toyo Spinning Co., Ltd.) were used as described above. Under the same conditions as in (2), PCR reaction, transformation of Escherichia coli JM109 strain, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies were performed.
As a result, pKK223-3-CFP-T7-261W / 263S, a double variant in which tyrosine at position 261 was replaced with tryptophan and glycine at position 263 was replaced with serine, was added to arginine at position 62. PKK223-3-CFP-T7-62S / 261W / 263S, which is a triple variant in which serine is replaced, tyrosine at position 261 is replaced with tryptophan, and glycine at position 263 is replaced with serine, and tyrosine at position 261 PKK223-3-CFP-T7-261W / 263S / 355H, tyrosine at position 261 is a triple variant in which glycine at position 263 is replaced with serine and alanine at position 355 is replaced with histidine. PKK223-3-CFP-T7-261W / 263S / 355D, arginine at position 62, which is a triple variant in which glycine at position 263 is replaced with serine and alanine at position 355 is replaced with aspartic acid. Is replaced with serine, tyrosine at position 261 is replaced with tryptophan, glycine at position 263 is replaced with serine, and alanine at position 355 is replaced with histidine, pKK223-3-CFP-T7. -62S / 261W / 263S / 355H, arginine at position 62 was replaced with serine, tyrosine at position 261 was replaced with tryptophan, glycine at position 263 was replaced with serine, and alanine at position 355 was replaced with asparagic acid. A quadruple variant, pKK223-3-CFP-T7-62S / 261W / 263S / 355D, was obtained.
Then, an Escherichia coli JM109 strain producing a modified amadriase in which a plurality of amino acid substitutions described in the column of "amino acid mutation" in Table 2 was introduced into the amino acid sequence shown in SEQ ID NO: 1 was obtained.
The Escherichia coli JM109 strain having the modified amadriase-producing ability obtained as described above was cultured by the method described in (3) above to prepare 0.6 ml of a crude enzyme solution of various modified amadriase.
Using the above-mentioned enzyme solution, the enzyme activity of αFL, αFA, αFG or αFV with respect to αFVH was measured by the method shown in the above-mentioned B: activity measurement method. For the activity measurement, a reagent 1: peroxidase and 4-aminoantipyrine solution adjusted to pH 7.0 were used. The results are shown in Table 2. In the data in the table, the upper value is the ratio of the reactivity of various glycated amino acids to the reactivity of αFVH, and the lower value is the reactivity after the introduction of mutation when the reactivity of CFP-T7 is 100. Is the ratio of.

That is, as shown in Table 2, in most cases, the substrate specificity was remarkably improved in the various multiple mutants prepared by combining the various single mutations confirmed in Example 1.
Specifically, αFL / αFVH in the double mutant in which tyrosine at position 261 was replaced with tryptophan and glycine at position 263 was replaced with serine was obtained when αFL / αFVH of CFP-T7 was 100%. On the other hand, it was 17%, αFA / αFVH was 30% when αFA / αFVH of CFP-T7 was set to 100%, and αFG / αFVH was set to 100% of αFG / αFVH of CFP-T7. It was 25% with respect to the case, and αFV / αFVH was 44% with respect to the case where αFV / αFVH of CFP-T7 was 100%.
Similarly, αFL / αFVH was 7% and αFA / αFVH in the triple mutant in which arginine at position 62 was replaced with serine, tyrosine at position 261 was replaced with tryptophan, and glycine at position 263 was replaced with serine. αFVH was 5%, αFG / αFVH was 6%, and αFV / αFVH was 17%.
Similarly, arginine at position 62, which has accumulated mutations in the triple mutant, is replaced with serine, tyrosine at position 261 is replaced with tryptophan, glycine at position 263 is replaced with serine, and position 355. In the quadruplex in which alanine was replaced with aspartic acid, αFL / αFVH was 2%, αFA / αFVH was 2%, αFG / αFVH was 0%, αFV / αFVH was 6%, and CFP. -Significant improvement compared to T7.
That is, the substrate specificity of the obtained further multiple mutants is gradually improved, and by appropriately combining the mutation points of the present invention confirmed in Example 1, an amadriase having further excellent substrate specificity can be obtained. It became clear that it could be created.

[Example 3]
(Reduction of reactivity to various glycated amino acids in the amadriase of the present invention)
Next, using the modified amadriane, saccharified amino acids other than αFL, αFA, αFG, and αFV for the reactivity of αFVH, that is, αFI, α-fructosylthreonine (αFT), α-fructosylmethionine (αFM), α- The reactivity of fructosylphenylalanine (αFF), α-fructosylserine (αFS), α-fructosylglutamic acid (αFE), εFK, β-fructosylalanine (βFA) was also evaluated.
D. : Preparation of Reagents Reagent 5: Amadriase solution The arginine at position 62 of the amadriase shown in SEQ ID NO: 1 and the amaliase shown in SEQ ID NO: 1 is replaced with serine, the tyrosine at position 261 is replaced with tryptophan, and the glycine at position 263 is serine. A modified amadriase (hereinafter referred to as CFP-T7-62S / 261W / 263S / 355D) in which alanine at position 355 was replaced with aspartic acid was prepared in the same manner as in Example 1 (3).
Reagent 6: Substrate solution (60 mM; final concentration 2 mM)
αFVH 250 mg, αFL 176 mg, αFA 151 mg, αFG 142 mg, αFV 167 mg, αFI 176 mg, αFT 169 mg, αFF 196 mg, αFM 187 mg, αFS 160 mg, αFE 185 mg, εFK 185 mg or βFA 185 mg or βFA To do.

In the above B: activity measurement method, the enzyme activity of various glycated amino acids with respect to αFVH was measured by a method using reagent 5 instead of reagent solution and reagent 6 instead of reagent 3. For the activity measurement, a reagent 1: peroxidase and 4-aminoantipyrine solution adjusted to pH 6.5 were used.
From the activity values measured as described above, αFL / αFVH corresponding to the activity value of αFL when the activity value of αFVH was set to 1 was calculated. Similarly for αFA, αFV, αFG, αFI, αFT, αFF, αFS, αFM, αFE, εFK, or βFA, αFA / αFVH, αFV / αFVH, αFG / αFVH, αFI / αFVH, αFT / αFVH, αFF / αFVH, αFS / αFVH, αFM / αFVH, αFE / αFVH, εFK / αFVH or βFA / αFVH were calculated. CFP-T7 was used as a comparative example, and CFP-T7-62S / 261W / 263S / 355D was used as an example. In addition, the ratio of the reactivity after the introduction of the mutation (ratio 2) to the ratio of the reactivity before the introduction of the mutation (ratio 1) of the present invention was calculated (ratio 2 / ratio 1). The above results are shown in Table 3.

As shown in Table 3, under the conditions of this example, the αFL / αFVH of CFP-T7 was 95%. In contrast, arginine at position 62 of CFP-T7 was replaced with serine, tyrosine at position 261 was replaced with tryptophan, glycine at position 263 was replaced with serine, and alanine at position 355 was replaced with aspartic acid. The αFL / αFVH of the modified amadrianase was 3%, which was reduced to 0.03 as compared with CFP-T7, that is, the αFL / αFVH could be reduced by 97%, and the substrate specificity was significantly improved. ..
Furthermore, the αFA / αFVH of CFP-T7 was 151%, and the αFA / αFVH of the modified amadriase was 2%, which was reduced to 0.01 compared to CFP-T7, that is, 99% of αFA / αFVH. Was able to be reduced, and the substrate specificity was significantly improved.
Furthermore, the αFV / αFVH of CFP-T7 was 225%, and the αFV / αFVH of the modified amadriase was 7%, which was reduced to 0.03 compared to CFP-T7, that is, 97% was also αFV / αFVH. Was able to be reduced, and the substrate specificity was significantly improved.
Furthermore, the αFI / αFVH of CFP-T7 was 269%, and the αFI / αFVH of the modified amadriase was 8%, which was reduced to 0.05 compared to CFP-T7, that is, 95% of αFI / αFVH. Was able to be reduced, and the substrate specificity was significantly improved.
Furthermore, the αFM / αFVH of CFP-T7 was 244%, and the αFM / αFVH of the modified amadriase was 4%, which was reduced to 0.02 compared to CFP-T7, that is, 98% was also αFM / αFVH. Was able to be reduced, and the substrate specificity was significantly improved.
Reactivity to saccharified amino acids other than αFL, αFA, αFV, αFI, αFM, specifically αFG, αFT, αFF, αFS, αFE, εFK, or βFA is reduced to at least 0.5, which is more remarkable. The most prominent ones decreased to 0.29, the more prominent ones decreased to 0.14, and the more prominent ones decreased to 0.04. That is, when the reactivity ratio before modification is 100%, the reactivity ratio after modification is reduced to at least 50%, the more remarkable one is reduced to 71%, and the more remarkable one is 86. It decreased to%, and more remarkable one decreased to 96%, and the substrate specificity was improved.
Based on the above, arginine at position 62 of CFP-T7 was replaced with serine, tyrosine at position 261 was replaced with tryptophan, glycine at position 263 was replaced with serine, and alanine at position 355 was replaced with aspartic acid. It was suggested that the type of amadriase can reduce the influence of glycated amino acids and react specifically with αFVH.

[Example 4]
(Measurement of fructosyl peptide sample in which αFV, αFL or εFK coexist)
(5) Preparation of fructosyl peptide sample containing αFV, αFL or εFK The solution of αFVH or αFV described in (Reagent 7) is mixed and diluted with ion-exchanged water to obtain a 0.6 mM αFVH solution, 0. A 6 mM αFV solution, a 0.12 mM αFV solution, a 0.06 mM αFV solution, a 0.6 mM αFL solution, a 0.12 mM αFL solution, and a 0.6 mM εFK solution were prepared.
By mixing an equal amount of ion-exchanged water, 0.6 mM αFV solution, 0.6 mM αFL solution, or 0.6 mM εFK solution with 0.6 mM αFVH solution, 0.3 mM αFVH solution or 0.3 mM αFV A mixed solution with, a mixed solution with 0.3 mM αFL, or a mixed solution with 0.3 mM εFK was used.
Similarly, by mixing 0.12 mM αFV solution, 0.06 mM αFV solution, or 0.12 mM αFL solution with 0.6 mM αFVH solution, a mixed solution of 0.3 mM αFVH solution and 0.06 mM αFV, Alternatively, a mixed solution with 0.03 mM αFV or a mixed solution with 0.06 mM αFL was used.

(6) Measurement of fructosyl peptide sample containing αFV, αFL or εFK (Reagent 7)
Amadriase solution comparative example: 5.0 U / ml CFP-T7
Example: 21.0U / ml CFP-T7-62S / 261W / 263S / 355D
(Reagent 8)
6.3 U / ml peroxidase (manufactured by Kikkoman Biochemifa)
0.62 mM 4-aminoantipyrine (manufactured by Wako Pure Chemical Industries, Ltd.)
0.7 mM TOOS (manufactured by Dojin Chemical Research Institute)
126 mM phosphate buffer (pH 6.5)

150 μl (Reagent 8) was added to 25 μl (Reagent 7), and the mixture was heated at 37 ° C. for 5 minutes. Next, 35 μl of the fructosyl peptide sample prepared in (5) above or a mixed sample with αFV, αFL or εFK was added to start the reaction, and the absorbance at a wavelength of 545 nm after the reaction at 37 ° C. for 5 minutes was measured by the automatic analyzer Bio. The sample was measured with Majesty JCA-BM1650 (JEOL Ltd.). Using ion-exchanged water instead of the sample and measuring the absorbance at a wavelength of 545 nm (reagent blank) as a control, the amount of change in absorbance when each sample was measured was calculated by the following formula.

Absorbance change = ΔAes-Ae0
ΔAes: Absorbance 5 minutes after the start of the reaction ΔAe0: Absorbance 5 minutes after the start of the reaction when the control solution was added

When the amount of change in absorbance when measuring a sample containing only 0.3 mM αFVH was set to 1 using each amount of change in absorbance obtained, a sample in which saccharified amino acids other than αFVH were mixed in addition to αFVH was measured. The ratio of the amount of change in absorbance was determined.
Specifically, first, the amount of change in absorbance when a sample containing only 0.3 mM αFVH was measured using CFP-T7 was determined. In addition, the amount of change in absorbance when a mixed sample containing 0.3 mM αFVH and 0.3 mM, 0.06 mM or 0.03 mM αFV was measured was determined. Then, when the amount of change in absorbance when measuring a sample containing only 0.3 mM αFVH is 1, it is the ratio of the amount of change in absorbance when measuring a 0.3 mM, 0.06 mM or 0.03 mM αFV mixed sample. “ΔA (αFVH + αFV) / ΔAαFVH” was calculated.

Similarly, the amount of change in absorbance when measuring a mixed sample containing 0.3 mM αFVH or 0.3 mM, 0.06 mM αFL was determined, and the amount of change in absorbance when measuring a sample containing only 0.3 mM αFVH was set to 1. “ΔA (αFVH + αFL) / ΔAαFVH”, which is the ratio of the amount of change in absorbance when the 0.3 mM or 0.06 mM αFL mixed sample was measured, was calculated.
Similarly, the amount of change in absorbance when a mixed sample containing 0.3 mM αFVH and 0.3 mM εFK was measured was obtained, and 0 when the amount of change in absorbance when a sample containing only 0.3 mM αFVH was measured was 1. .. “ΔA (αFVH + εFK) / ΔAαFVH”, which is the ratio of the amount of change in absorbance when the 3 mM εFK mixed sample was measured, was calculated.
On the other hand, instead of CFP-T7, CFP-T7-62S / 261W / 263S / 355D was used to measure each of the above-mentioned samples in the same manner, and from the amount of change in absorbance, "ΔA (αFVH + αFV) / ΔAαFVH" and " ΔA (αFVH + αFL) / ΔAαFVH ”and“ ΔA (αFVH + εFK) / ΔAαFVH ”were calculated. Then, for each, both were compared. The results are shown in Table 4. In the table, the numerical values in the third column from the right are the four columns from the right in addition to αFVH when the amount of change in absorbance when a sample containing only 0.3 mM αFVH is measured using CFP-T7. It is the ratio of the amount of change in absorbance when the sample mixed with the saccharified amino acids described in the eyes is measured. In the table, the values in the second column from the right are the samples containing only 0.3 mM αFVH when each sample was measured in the same manner using CFP-T7-62S / 261W / 263S / 355D. It is the ratio of the amount of change in absorbance when the sample in which the saccharified amino acid described in the fourth column from the right is mixed in addition to αFVH is measured when the amount of change in absorbance at time is 1.

As is clear from Table 4, "ΔA (αFVH + αFV) / ΔAαFVH", "ΔA (αFVH + αFL) / ΔAαFVH", and "ΔA (αFV + εFK)" measured using reagents containing CFP-T7-62S / 261W / 263S / 355D. / ΔAαFVH ”was all lower than the values obtained using the reagent containing CFP-T7 before introducing the substitution of the present invention. That is, it was confirmed that by introducing the substitution of the present invention, the influence on the measured values of the saccharified amino acids other than αFVH to be mixed was reduced.
In the rightmost column of Table 4, the degree to which the above-mentioned "effect on the measured values of glycated amino acids other than αFVH to be mixed is reduced" is shown as "effect reduction rate (%) of saccharified amino acids". This numerical value is calculated by calculating the difference (decrease) between the numerical value in the third column from the right and the numerical value in the second column from the right, and dividing this by the numerical value in the third column from the right.
Specifically, when CFP-T7-62S / 261W / 263S / 355D is used instead of CFP-T7, the measured value is higher when 0.3 mM αFL is mixed than when only αFVH is measured. The degree of appearance decreased by 53%. It was also found that when 0.06 mM αFL was mixed, it decreased by 24%. Also, when 0.3 mM αFV is mixed, the effect of αFV is reduced by 41%, when 0.06 mM αFV is mixed, it is reduced by 18%, and when 0.03 mM αFV is mixed, it is reduced by 9%. It turned out. Similarly, it was found that when CFP-T7-62S / 261W / 263S / 355D was used rather than CFP-T7, the effect of εFK was reduced by 8% when 0.3 mM εFK was contained.

Further, from Table 4, when 0.06 mM αFL or 0.03 mM αFV is mixed, that is, when αFL having a final concentration of 10 μM or αFV having a final concentration of 5 μM coexists, CFP-T7-62S / 261W / 263S It was found that by using / 355D, the influence of coexisting αFL or 5 μM αFV was suppressed within 5%, and αFVH could be measured accurately.
According to Biological Sample Analysis Vol27, p97-103, 2004, the amount of glycated albumin contained in the blood of patients who received Unicalic (registered trademark), which is a high-calorie amino acid infusion, was measured by an enzymatic method. It has been shown that the value obtained by dividing by the amount of serum albumin is on average 6.88% higher than the value measured by the HPLC method. Based on this finding, when the serum albumin amount was 3.8 g / dL and the molecular weight of albumin was 66,000, administration of Unicalic (registered trademark) increased the blood glycated amino acid concentration by 39.4 μM. It is thought that. In addition, since Unicalic (registered trademark) contains 12.9% L-leucine and 9.6% L-valine, it is derived from a high-calorie amino acid infusion, assuming that all of these are saccharified. Of the 39.4 μM glycated amino acids mixed into the body, αFL contains 5 μM and αFV contains 4 μM.
For example, when the effect of CFP-T7-62S / 261W / 263S / 355D is estimated based on the above values, CFP-T7-62S / 261W / 263S / 355D is 10 μM αFL or 5 μM. Even if αFV coexists, it is considered that αFVH can be measured accurately with almost no effect, and it may be affected by glycated amino acids that are predicted to be brought into the blood of patients who received Unicalic®. It is suggested that the sex is significantly lower. That is, it was found that by using the substitution-introduced amadriase of the present invention, αFVH can be easily and accurately quantified without being affected by the glycated amino acid derived from the high-calorie amino acid infusion solution, which has been a problem in the past.

[Example 5]
(Introduction of point mutations into various amadriase)
The findings of the present invention were first verified using amadriase derived from the genus Coniochaeta, but as described above, the amino acid sequence of amadriase derived from other species with reference to the information obtained by the known alignment treatment based on sequence identity. By introducing a similar mutation at the corresponding position in, it can be suggested that a similar effect can be expected. Therefore, the inventors actually introduced the findings of the present invention into a plurality of amadriase other than the amadriase derived from the genus Coniochaeta, and carried out this verification.

1. 1. Introducing a point mutation into a ketoamine oxidase gene derived from Neocospospora plasmid SEQ ID NO: 30 is an amino acid sequence of ketoamine oxidase derived from Neocospospora vasinfecta, and is a recombinant into which a gene encoding the amino acid sequence of SEQ ID NO: 30 (SEQ ID NO: 36) is inserted. By expressing the plasmid pET22b-NvFX in Escherichia coli, the activity of ketoamine oxidase derived from Neocosmospora vasinfecta has been confirmed (see International Publication No. 2012/18094).

Synthetic oligonucleotides of SEQ ID NOs: 37 and 38, KOD-Plus- (manufactured by Toyo Spinning Co., Ltd.), using the recombinant plasmid pET22b-NvFX as a template, in order to introduce a substrate specificity-improving mutation into ketoamine oxidase derived from Neocosmospora vasinfecta. ) Was used for PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of the DNA encoding amadriase in the plasmid DNA retained by the growing colony under the same conditions as in Example 1. As a result, a recombinant plasmid (pET22b-NvFX-62S) encoding a ketoamine oxidase gene derived from Neocosmospora vasinfecta in which arginine at position 62 of the amino acid sequence shown in SEQ ID NO: 30 was replaced with serine was obtained.
Then, Escherichia coli BL21 (DE3) strain was transformed under the same conditions as in Example 1 to obtain Escherichia coli BL21 (DE3) (pET22b-NvFX-62S) strain.

2. 2. Introducing a point mutation into the fructosyl amino acid oxidase gene derived from Pencillium crysogenum SEQ ID NO: 35 is an amino acid sequence of fructosyl amino acid oxidase derived from Pencillium crysogenum in which serine at position 60 is replaced with glycine to impart fructosyl peptide oxidase activity. By expressing in Escherichia coli the recombinant plasmid pET22b-PcFX into which the gene encoding the amino acid sequence of SEQ ID NO: 35 has been inserted, the activity of fructosyl amino acid oxidase derived from Pencillium lysogenum has been confirmed (International Publication No. 2012/18094). See brochure).

KOD-Plus- (Toyo Spinning Co., Ltd.), a synthetic oligonucleotide of SEQ ID NOs: 40 and 41, using the recombinant plasmid pET22b-PcFX as a template in order to introduce a substrate-specificity-improving mutation into fructosyl amino acid oxidase derived from Pencillium crysogenum. The same conditions as in Example 1 were used to carry out PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of the DNA encoding amadriase in the plasmid DNA retained by the growing colony. As a result, a recombinant plasmid (pET22b-PcFX-62S) encoding a fructosyl amino acid oxidase gene derived from Pencillium crysogenum in which arginine at position 62 of the amino acid sequence shown in SEQ ID NO: 35 was replaced with serine was obtained.
Then, Escherichia coli BL21 (DE3) strain was transformed under the same conditions as in Example 1 to obtain Escherichia coli BL21 (DE3) (pET22b-PcFX-62S) strain.

3. 3. Introducing a point mutation into the Aspergillus nidulans-derived fructosyl amino acid oxidase gene SEQ ID NO: 33 is an amino acid sequence of Aspergillus nidulans-derived fructosyl amino acid oxidase in which serine at position 59 is replaced with glycine in order to impart fructosyl peptide oxidase activity. The activity of Aspergillus nidulans-derived fructosyl amino acid oxidase has been confirmed by expressing in Escherichia coli the recombinant plasmid pET22b-AnFX into which the gene encoding the amino acid sequence of SEQ ID NO: 33 has been inserted (International Publication No. 2012/18094). See brochure).

In order to introduce a substrate-specificity-improving mutation into fructosyl amino acid oxidase derived from Aspergillus nidulans, a synthetic oligonucleotide of SEQ ID NOs: 43 and 44, KOD-Plus- (Toyo Spinning Co., Ltd.) was used as a template using the recombinant plasmid pET22b-AnFX. The same conditions as in Example 1 were used for PCR reaction, transformation of Escherichia coli JM109, and nucleotide sequence determination of DNA encoding amadriase in plasmid DNA retained by growing colonies. As a result, a recombinant plasmid (pET22b-AnFX-61S) encoding a fructosyl amino acid oxidase gene derived from Aspergillus nidulans in which arginine at position 61 of the amino acid sequence shown in SEQ ID NO: 33 was replaced with serine was obtained.
Then, Escherichia coli BL21 (DE3) strain was transformed under the same conditions as in Example 1 to obtain Escherichia coli BL21 (DE3) (pET22b-AnFX-61S) strain.

4. Evaluation of Substrate Specificity Improving Effect of Various Amadriase Introduced Point Mutations Escherichia coli BL21 (DE3) (pET22b-NvFX-62S) strain, Escherichia coli BL21 (DE3) (DE3) having the modified amadriase-producing ability obtained as described above. The pET22b-PcFX-62S) strain and the Escherichia coli BL21 (DE3) (pET22b-AnFX-61S) strain were cultured by the method of Example 1 to prepare 0.6 ml of a crude enzyme solution of various modified amadriase. For comparison, Escherichia coli BL21 (DE3) (pET22b-NvFX) strain, Escherichia coli BL21 (DE3) (pET22b-PcFX) strain, Escherichia coli BL21 (DE3) (pET22b-AnFX) strain having amadriase-producing ability before introduction of the mutation Was also cultured in the same manner to prepare 0.6 ml of crude enzyme solution of various amadriase. Using each of the obtained crude enzyme solutions as a sample, the substrate specificity of various modified amadriase was evaluated according to the activity measurement method according to Example 1 (4). The results are shown in Table 5. In the data in the table, the upper value is the ratio of the reactivity of various glycated amino acids to the reactivity of αFVH, and the lower value is the reaction after the introduction of the mutation when the reactivity of various amadriase before modification is 100. The ratio of sex.

As shown in Table 5, under the conditions of this example, αFL / αFVH of NvFX-62S decreased to 91% as compared with the case where αFL / αFVH of NvFX was set to 100%. Similarly, αFA / αFVH decreased to 64%, αFG / αFVH decreased to 68%, and αFV / αFVH decreased to 78%.
Further, the αFL / αFVH of PcFX-62S decreased to 81% with respect to the case where αFL / αFVH of PcFX was set to 100%. Similarly, αFA / αFVH decreased to 86%, αFG / αFVH decreased to 36%, and αFV / αFVH decreased to 87%.
Further, the αFL / αFVH of AnFX-61S decreased to 67% with respect to the case where the αFL / αFVH of AnFX was 100%. Similarly, αFA / αFVH decreased to 21%, αFG / αFVH decreased to 30%, and αFV / αFVH decreased to 63%.
That is, the substitution of the amino acid at the position corresponding to arginine at position 62 of the Amadriase derived from the genus Coniochaeta shown in SEQ ID NO: 1 with serine is a ketoamine oxidase derived from Neocosmospora vasinfecta, a fructosyl amino acid oxidase derived from Penicillium crysogenum, and Aspergillus. It was confirmed that amino acid oxidase is also an effective substitution for the production of amadriase whose substrate specificity is actually improved.

Claims (8)

The following (i) and (ii):
(I) It has an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 30, 33 or 35;
(Ii) Substitution:
(V) the amino acid positions corresponding to position 64 of SEQ ID NO: 1, serine, the substitution of aspartic acid or glutamic acid;
The activity was measured at a substrate final concentration of 5 mM and pH 7.0, and the reactivity to α-fructosylvalyl histidine was measured.
From ( b ) to (d) below:
( B) α-fructosylalanine (c) α-fructosylglycine (d) The percentage of reactivity to one or more glycated amino acids selected from the group consisting of α-fructosylvaline does not have the above substitutions. Amadriase with reduced properties compared to amadriase. (V) the amino acid position corresponding to position 64 of SEQ ID NO: 1 is substituted with serine;
The amadriase according to claim 1. Amadriase, and more
It follows from (t) (z):
(T) amino acid at the position corresponding to position 62 of SEQ ID NO: 1 is replaced with alanine or serine;
(U) amino acid at the position corresponding to position 63 of SEQ ID NO: 1 is substituted with alanine;
(W) an amino acid in the position corresponding to 261 of SEQ ID NO: 1 is substituted phenylalanine, aspartic or script fan;
(Z) the amino acid at the position corresponding to 417 of SEQ ID NO: 1 is substituted with histidine;
The amadriase according to claim 1 or 2 , which has any one or more of the above . The amadriase gene encoding the amino acid sequence according to any one of claims 1 to 3 . A recombinant vector containing the amadriase gene according to claim 4 . A host cell containing the recombinant vector according to claim 5 . The following steps:
(I) The step of culturing the host cell according to claim 6 ;
(Ii) A method for producing amadriase, which comprises a step of expressing an amadriase gene contained in a host cell; and (iii) a step of isolating the amadriase from a culture. A kit for measuring glycated hemoglobin, which comprises the amadriase according to any one of claims 1 to 3 .

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