JP5465415B2 - Fructosyl amino acid measuring enzyme and its use - Google Patents

Description

  The present invention relates to a protein having fructosyl amino acid oxidase activity having excellent characteristics in the measurement of fructosyl amino acids. More specifically, it is a mutant protein having fructosyl amino acid oxidase activity and having a ratio of ε-fructosyl-L-lysine reactivity to fructosyl-L-valine reactivity lower than that of the wild type. The present invention also relates to a method for using the mutant protein, such as a method for measuring fructosyl amino acids, a kit for measuring glycated amino acids, a sensor for measuring glycated amino acids, and the like.

  As a blood glucose control marker for diabetic patients, measurement of hemoglobin A1c (HbA1c) and glycoalbumin, which are glycated proteins contained in blood proteins, is heavily used. The glycated protein is produced by a reaction between D-glucose present in blood and amino acid residues constituting the blood protein. The main glycation sites of blood proteins are the ε-amino group of lysine residues and the α-amino group of the amino terminal amino acid of blood proteins. When measuring HbA1c, the amount of glycated protein produced by binding D-glucose to the α-amino group of valine, which is the amino terminal amino acid of hemoglobin β chain, is measured.

  In recent years, enzymatic measurement methods capable of measuring glycated proteins in blood easily and in a short time have been developed and already commercialized. By utilizing the enzymatic measurement method, it is possible to measure glycated protein with high throughput, which is useful in the clinical laboratory field. In the enzymatic method, first, a glycated protein is hydrolyzed with a protease, and then generated fructosyl-L-valine (hereinafter also referred to as “F-Val”), ε-fructosyl-L-lysine (hereinafter referred to as “F-”). Glycated amino acids, such as fructosyl amino acids, are also oxidatively hydrolyzed with fructosyl amino acid oxidase (hereinafter also referred to as “FAOD”). Finally, the hydrogen peroxide generated by the oxidase reaction is colorimetrically determined by a peroxidase-chromogen reaction system (see Patent Documents 1 to 11).

In the measurement of glycated protein by the enzymatic method, the substrate specificity of FAOD, which is the main reaction enzyme, is an important factor. For example, in the measurement of HbA1c, an enzyme with high specificity for F-Val is desired. In addition, an enzyme that acts on fructosyl valyl histidine is desired in order to perform measurement specific to the β chain of glycated hemoglobin. This is because the amino terminal amino acids of hemoglobin α-chain and β-chain are both valine. Therefore, in order to perform measurement specific to β-chain, two amino-terminal amino acid residues (that is, fructosylvalylhistidine) This is because recognition is necessary. (See Patent Documents 12 to 14, Non-Patent Document 1, and Non-Patent Document 2.)
JP 2004-129531 A (publication date: April 30, 2004 (2004. 4.30)) JP 2004-1113014 A (publication date: April 15, 2004 (2004.4.15)) International Publication No. 2003/064683 Pamphlet (International Publication Date: August 7, 2003 (2003.8.7)) JP 2007-181466 A (publication date: July 19, 2007 (2007.7.19)) JP 2007-289202 A (publication date: November 8, 2007 (2007. 11.8)) JP 2005-110657 A (publication date: April 28, 2005 (2005. 4.28)) Japanese Patent No. 4045322 (issue date: February 13, 2008 (2008.2.13)) Japanese Patent No. 4014088 (issue date: September 21, 2007 (2007.9.21)) Japanese Patent No. 4039664 (issue date: January 30, 2008 (2008.1.30)) Japanese Patent No. 4010474 (issue date: November 21, 2007 (2007.11.21)) Japanese Patent No. 3971702 (issue date: September 5, 2007 (2007.9.5)) International Publication No. 2005/049857 (International Publication Date: June 2, 2005 (2005.6.2)) International Publication No. 2005/049858 (International Publication Date: June 2, 2005 (2005.6.2)) JP 2003-235585 A (publication date: August 26, 2003 (2003.8.26)) Arch. Microbiol., 180: 227-231 (2003) Biochem. Biophys. Res. Commun., 311: 104-111 (2003)

  However, the α-amino group in the protein chain is one amino terminal amino acid residue, whereas there are usually many lysine residues that can be F-Lys in the protein chain. For this reason, even if the FAOD used in the measurement of HbA1c has a high substrate specificity for F-Val, it is difficult to accurately measure HbA1c if the reactivity to F-Lys is high.

  Therefore, as a protein having FAOD activity suitable for measurement of glycated protein, a mutant protein having a lower ratio of F-Lys reactivity to F-Val reactivity than that of the wild type is desired.

  The present invention has been made in view of the above problems, and an object thereof is a protein having fructosyl amino acid oxidase activity useful for measurement of fructosyl amino acids, and ε for fructosyl-L-valine reactivity. -To create and provide a mutant protein with a reduced fraction of fructosyl-L-lysine reactivity than the wild type. In addition, as a typical application example of the mutant protein, a fructosyl amino acid measurement method using the mutant protein, a glycated protein measurement kit, a glycated protein sensor, and the like are provided.

  As a result of intensive investigations to achieve the above object, the present inventors have substituted ε- for fructosyl-L-valine reactivity by substituting some amino acid residues of a protein having fructosyl amino acid oxidase activity. We succeeded in constructing a mutant protein in which the fraction of fructosyl-L-lysine reactivity was lower than that of the wild type. Furthermore, the present inventors confirmed that fructosyl amino acids can be measured by allowing the protein to act on a fructosyl amino acid-containing specimen and quantifying the resulting hydrogen peroxide by a peroxidase reaction. The invention has been completed. That is, the present invention includes the following inventions.

The protein according to the present invention is a protein characterized by the following (I) or (II):
(I) one or more amino acid residues of 55th asparagine, 268th phenylalanine, 286th phenylalanine, and 349th cysteine from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 are substituted; A protein having fructosyl amino acid oxidase activity and having a reduced ratio of ε-fructosyl-L-lysine reactivity to fructosyl-L-valine reactivity compared to wild-type protein;
(II) In the protein of (I), one or several amino acid residues are substituted, deleted, and / or added, and ε-fructosyl-L-lysine reactive to fructosyl-L-valine reactivity Proteins with a reduced proportion of wild type protein.

  The protein of the present invention is a protein in which the 55th asparagine from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with one amino acid selected from the group consisting of glycine, valine, serine, and threonine It is preferable.

  In the protein of the present invention, the 268th phenylalanine from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 is selected from the group consisting of tryptophan, alanine, serine, threonine, valine, leucine, methionine, cysteine, and histidine. A protein substituted with one amino acid is preferred.

  The protein of the present invention is a protein in which the 286th phenylalanine from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with one amino acid selected from the group consisting of methionine, valine, and leucine. preferable.

The protein according to the present invention may be a protein characterized by any of the following (a) to (e):
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
(B) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5;
(C) a protein consisting of the amino acid sequence shown in SEQ ID NO: 7;
(D) a protein consisting of the amino acid sequence shown in SEQ ID NO: 9;
(E) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the proteins (a) to (d).

  The protein consisting of the amino acid sequence represented by SEQ ID NO: 1 may be fructosyl amino acid oxidase derived from Aspergillus nidulans.

  The present invention also includes a polynucleotide encoding the protein according to the present invention.

  A polynucleotide according to the present invention is a polynucleotide having the base sequence shown in SEQ ID NO: 4, a polynucleotide having the base sequence shown in SEQ ID NO: 6, a polynucleotide having the base sequence shown in SEQ ID NO: 8, or a sequence The polynucleotide which has a base sequence shown by number 10 may be sufficient. In addition, polynucleotides that hybridize with the above-mentioned polynucleotides under stringent conditions are also included in the present invention.

  The present invention also includes a vector comprising the polynucleotide according to the present invention.

  The present invention also includes a transformant transformed with the vector according to the present invention.

  Moreover, this invention includes the manufacturing method of fructosyl amino acid oxidase including the process of culture | cultivating the transformant concerning the said invention.

  Moreover, this invention includes the measuring method of a fructosyl amino acid including the process which makes the protein concerning this invention act on glycated amine.

  The present invention also includes a glycated protein measurement kit comprising at least the protein according to the present invention.

  The present invention also includes a glycated protein sensor comprising at least the protein according to the present invention.

  It is well known to those skilled in the art that even if the three-dimensional structure of a protein has been determined, it is difficult to rationally improve the substrate specificity. Furthermore, until now, the determination of the three-dimensional structure of fructosyl amino acid oxidase has not been made, and it has been widely known to those skilled in the art that it is very difficult to obtain an indicator of mutant protein construction itself. The present invention has been completed by overcoming the technical difficulties described above through the diligent efforts of the inventors. Therefore, it can be said that the present invention has a remarkable and advantageous effect that cannot be expected by those skilled in the art.

  According to the present invention, it is possible to provide a protein having fructosyl amino acid oxidase activity, which has excellent properties for measuring fructosyl amino acids, and a method for using the same.

  An embodiment of the present invention will be described in detail as follows, but the present invention is not limited to this. In addition, all the nonpatent literatures and patent literatures described in this specification are used as reference in this specification. In addition, “˜” in the present specification means “above and below”. For example, if “★ to ☆” is described in the specification, it means “★ or more and ☆ below”. In the present specification, “and / or” means either one or both.

<1. Protein according to the present invention>
The protein according to the present invention is a protein characterized by the following (I) or (II).

(I) one or more amino acid residues of 55th asparagine, 268th phenylalanine, 286th phenylalanine, and 349th cysteine from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 are substituted; A protein having fructosyl amino acid oxidase activity and having a reduced ratio of ε-fructosyl-L-lysine reactivity to fructosyl-L-valine reactivity compared to wild-type protein;
(II) In the protein of (I), one or several amino acid residues are substituted, deleted, and / or added, and ε-fructosyl-L-lysine reactive to fructosyl-L-valine reactivity Proteins with a reduced proportion of wild type protein.

  The amino acid sequence shown in SEQ ID NO: 1 is a wild-type derived from the genome database of Aspergillus nidulans (http://www.broad.mit.edu/annotation/genome/aspergillus#group/GenomesIndex.html). Amino acid sequence of type FAOD.

  In the present invention, the “wild type protein” represents a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

  As long as it has fructosyl amino acid oxidase activity in the protein according to the present invention, the 55th asparagine, 268th phenylalanine, 286th from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 The amino acid residue is not particularly limited as long as it is any one or more amino acid residues of phenylalanine and 349th cysteine, and may be the four amino acid residues alone or a combination thereof. In addition, the amino acid which substitutes the amino acid of the said location is not specifically limited as long as the protein after substitution has fructosyl amino acid oxidase activity. For example, the 55th asparagine may be replaced with glycine, valine, serine or threonine. The 268th phenylalanine may be substituted with tryptophan, alanine, serine, threonine, valine, leucine, methionine, cysteine or histidine. The 286th phenylalanine may be substituted with methionine, valine or leucine. The 349th cysteine may be substituted with valine.

As an amino acid sequence of one Embodiment of the protein concerning this invention, the amino acid sequence in any one of the following (a)-(d) is mentioned, for example. Needless to say, the protein according to the present invention is not limited to the following.
(A) amino acid sequence shown in SEQ ID NO: 3 (b) amino acid sequence shown in SEQ ID NO: 5 (c) amino acid sequence shown in SEQ ID NO: 7 (d) amino acid sequence shown in SEQ ID NO: 9 The protein consisting of the amino acid sequence is a protein in which the 55th asparagine from the amino terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is substituted with glycine. The protein consisting of the amino acid sequence shown in SEQ ID NO: 5 is a protein in which tryptophan is substituted for the 268th phenylalanine from the amino terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1. The protein consisting of the amino acid sequence shown in SEQ ID NO: 7 is a protein in which 286th phenylalanine is substituted with methionine from the amino terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1. The protein consisting of the amino acid sequence shown in SEQ ID NO: 9 is a protein in which the 349th cysteine from the amino terminus of the protein having the amino acid sequence shown in SEQ ID NO: 1 is substituted with valine.

  The present invention also includes an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence of the protein according to the present invention described above, and has fructosyl amino acid oxidase activity. Including a protein, a site where one or several amino acids are deleted, substituted and / or added may be used if the protein after deletion, substitution and / or addition has fructosyl amino acid oxidase activity. Any site in the amino acid sequence may be used. Here, “one or several amino acid residues” specifically refers to the number of amino acid residues within a range of 10 or less.

  As used herein, “reactivity” refers to oxidase activity measured using a fructosyl amino acid as a substrate. The reactivity can be evaluated by various known methods such as an activity measurement method as described in Examples.

  In the present specification, “ratio of ε-fructosyl-L-lysine reactivity to fructosyl-L-valine reactivity” refers to ε-fructosyl-L-lysine as a substrate for oxidase activity using fructosyl-L-valine as a substrate. It represents the ratio of oxidase activity.

  The fructosyl amino acid oxidase activity in the present invention is measured by the method described in the “Activity Measurement Method” section of the Examples described later. When measuring fructosyl amino acid oxidase activity, a fructosyl amino acid such as F-Val or F-Lys may be used as a substrate. In the description of the present invention, “having fructosyl amino acid oxidase activity” preferably means 0.1 U / mg protein or more, more preferably 1.0 U / mg protein or more, using F-Val as a substrate. Means.

  The protein according to the present invention may be produced, for example, using a gene recombination technique, or may be chemically synthesized using an amino acid synthesizer or the like. Various recombinant protein expression systems suitably used in the genetic recombination technique may be, for example, an E. coli expression system, insect cell expression system, mammalian cell expression system, and cell-free expression system, but are not limited thereto. The method for producing the protein of the present invention will be described later.

  The protein and the like according to the present invention are, for example, those subjected to intermolecular and / or intramolecular crosslinking (for example, disulfide bond), and chemical modification (for example, sugar chain, phosphoric acid or other functional groups). However, the present invention is not particularly limited to these, and those provided with a label (for example, histidine tag) or those provided with a fusion protein (for example, streptavidin, cytochrome, and GFP). Furthermore, the protein and the like according to the present invention may include a chimeric protein constituted by combining several types of protein fragments as long as the fructosyl amino acid oxidase activity is substantially maintained.

  You may comprise a fructosyl amino acid oxidase agent from the protein concerning this invention, excipient | fillers, such as serum protein, organic acid, and dextran. The fructosyl amino acid oxidase agent may contain a known article as a constituent article of the enzyme agent.

<2. Polynucleotides and the like according to the present invention>
The polynucleotide according to the present invention is characterized by encoding the protein according to the present invention.

  Here, the polynucleotide may be present in the form of DNA (eg, cDNA or genomic DNA) or RNA (eg, mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand).

  Moreover, the polynucleotide concerning this invention may be chemically synthesize | combined and codon usage (Codon usage) may be changed so that the expression of the protein which codes may be improved.

  As a method for modifying a polynucleotide according to the present invention, a commonly used method for modifying a polynucleotide is used. That is, a polynucleotide having genetic information of a recombinant protein may be prepared by substituting, deleting, and / or adding a specific base of a polynucleotide having protein genetic information. Specific methods for converting the base of the polynucleotide include, for example, use of a commercially available kit (Transformer Site-Directed Mutagenesis Kit; manufactured by Clonetech, QuickChange Site Directed Mutagenic Kit; Stratagene, etc.) or polymerase chain reaction. Use. These methods are known to those skilled in the art.

  As long as the polynucleotide concerning this invention codes the protein concerning the said invention, the base sequence will not be specifically limited. Therefore, the present invention includes all polynucleotides having a base sequence corresponding to the amino acid sequence of the protein according to the present invention.

  As one embodiment of the polynucleotide according to the present invention, for example, a polynucleotide having the base sequence shown in SEQ ID NO: 4, a polynucleotide having the base sequence shown in SEQ ID NO: 6, and a base sequence shown in SEQ ID NO: 8 And a polynucleotide having the base sequence shown in SEQ ID NO: 10.

  Here, the base sequence shown in SEQ ID NO: 4 is a protein having an amino acid sequence in which the 55th asparagine of the amino acid sequence shown in SEQ ID NO: 1 is substituted with glycine (may be referred to as “N55G” for convenience). The base sequence to be encoded. The base sequence shown in SEQ ID NO: 6 is a base sequence encoding a protein having the amino acid sequence in which the 268th phenylalanine of the amino acid sequence shown in SEQ ID NO: 1 is substituted with tryptophan (may be referred to as “F268W” for convenience). It is. The base sequence shown in SEQ ID NO: 8 encodes a protein having an amino acid sequence in which the 286th phenylalanine in the amino acid sequence shown in SEQ ID NO: 1 is substituted with methionine (may be referred to as “F286M” for convenience). It is a base sequence. The base sequence shown in SEQ ID NO: 10 codes for a protein having an amino acid sequence in which the 349th cysteine of the amino acid sequence shown in SEQ ID NO: 1 is substituted with valine (may be referred to as “C349V” for convenience). It is a base sequence.

  In one embodiment of the present invention, the polynucleotide according to the present invention may be substituted with a chemically synthesized base. Moreover, the site | part by which the polynucleotide concerning this invention is substituted is not specifically limited, The protein expressed from the base sequence after substitution should just have a suitable property.

  Although the polynucleotide concerning this invention may consist only of the polynucleotide which codes the protein concerning this invention, the other base sequence may be added. The base sequence to be added is not limited, but includes a label (for example, histidine tag, Myc tag, and FLAG tag), a fusion protein (for example, GST and MBP), a promoter sequence (for example, yeast-derived promoter sequence, phage) Derived promoter sequences, E. coli derived promoter sequences, etc.), and base sequences encoding signal sequences (eg, endoplasmic reticulum translocation signal sequences, secretory sequences, etc.). The site to which these base sequences are added is not particularly limited, and may be the N-terminus or the C-terminus of the translated protein.

  The polynucleotide according to the present invention includes a polynucleotide encoding the protein according to the present invention (for example, a polynucleotide having the nucleotide sequence shown in SEQ ID NOs: 4, 6, 8, 10), or a complementary thereof. A polynucleotide that hybridizes with a polynucleotide comprising a base sequence under stringent conditions is also included.

  Here, the “stringent condition” refers to a condition in which nucleic acids having high homology, for example, hybridize at a temperature ranging from the Tm value of a perfectly matched hybrid to a temperature 15 ° C., preferably 10 ° C. lower. . As a specific example, it means a condition for hybridization in a general hybridization buffer at 68 ° C. for 20 hours.

  The base sequence of the polynucleotide according to the present invention can be determined by the dideoxy method described in Science, 214: 1205 (1981).

  On the other hand, the vector according to the present invention contains the polynucleotide according to the present invention. Other configurations are not particularly limited as long as they include the polynucleotide according to the present invention. As a vector serving as a base constituting the vector according to the present invention, a suitable vector in the host cell can be appropriately selected. When a plasmid vector is used as the vector according to the present invention, for example, pBluescript (registered trademark), pUC18 and the like can be used. In this case, examples of the host microorganism into which the vector is introduced include yeast, Escherichia coli (Escherichia coli W3110 (Escherichia coli), Escherichia coli C600, Escherichia coli JM109, Escherichia coli DH5α), insect cells, mammalian cells, and the like. Is available.

  The method for introducing the vector according to the present invention into the host microorganism is not particularly limited. For example, when the host microorganism introduces a vector into a microorganism belonging to the genus Escherichia, the recombinant DNA is present in the presence of calcium ions. A method of introducing selenium or a method of using electroporation can be applied. In addition, gene transfer may be performed using a commercially available competent cell (for example, competent high JM109, competent high DH5α; manufactured by Toyobo).

  In order to construct the vector according to the present invention, the polynucleotide according to the present invention is separated and purified, and then the fragment of the polynucleotide cleaved using a restriction enzyme treatment or the like and the base vector are cleaved with a restriction enzyme. The linear polynucleotide obtained in this manner can be constructed by closing the bond. For ligation and closure, DNA ligase or the like can be used depending on the nature of the vector and the polynucleotide. After introducing the vector of the present invention into a replicable host, screening is performed using the expression of the marker of the vector and the enzyme activity as an index, and a transformant containing the polynucleotide of the present invention can be obtained. Therefore, the vector according to the present invention preferably contains a marker gene such as a drug resistance gene.

  In addition, this invention includes the transformant transformed with the vector concerning the said invention. The host cell transformed with the vector according to the present invention is not particularly limited, and examples thereof include yeast, Escherichia coli, insect cells, and mammalian cells.

<3. Method for producing protein according to the present invention>
The method for producing a protein according to the present invention includes a step of culturing the transformant according to the present invention (referred to as “culturing step”). The method for producing a protein according to the present invention may include other steps that may be included in protein production using a transformant in addition to the above-described culturing step. Examples of other steps include a step of recovering the protein produced by the transformant according to the present invention after the culturing step and a step of purifying the protein.

(3-1) Culture process In the culture process, a large amount of recombinant protein can be stably produced by culturing the transformant according to the present invention in a nutrient medium. For the culture form of the transformant, the culture conditions may be selected in consideration of the nutritional physiological properties of the host. Industrially, aeration and agitation culture is advantageous.

  As a nutrient source of the nutrient medium used in the culturing step, those commonly used for culturing may be widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, lactose, molasses, and pyruvic acid are used. The nitrogen source may be any nitrogen compound that can be used, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract. In addition to these, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like may be added to the medium as necessary.

  The culture temperature of the transformant according to the present invention can be appropriately changed as long as the transformant is within a range capable of producing the protein according to the present invention. For example, when Escherichia coli is used as a host, it is preferable. Is about 20 to 42 ° C. The culture time may be completed at an appropriate time when the protein of the present invention reaches the maximum yield, and is usually about 6 to 48 hours. The pH of the medium can be appropriately changed within the range in which the transformant according to the present invention is suitably grown and the protein according to the present invention can be produced, but is preferably in the range of about pH 6.0 to 9.0. .

(3-2) Recovery Step When the transformant according to the present invention secretes the protein outside the cell, the culture contains the protein of the present invention. Therefore, the culture can be used as it is as the protein according to the present invention. At this time, you may isolate | separate a culture solution and the transformant concerning this invention by filtration, centrifugation, etc., for example.

  When the protein according to the present invention is present in the transformant, the transformant is collected from the culture obtained by culturing the transformant using means such as filtration or centrifugation, and the transformant is collected. The target protein may be recovered after disruption by a mechanical method or an enzymatic method such as lysozyme. If necessary, a chelating agent (for example, EDTA and the like) and a surfactant (for example, Triton-X100 and the like) may be added to solubilize the protein according to the present invention and separated and collected as an aqueous solution.

(3-3) Purification step The purification step is a step of purifying the protein obtained by the recovery step. Although the specific method of a refinement | purification process is not specifically limited, For example, the solution containing the protein concerning this invention is concentrate | evaporated under reduced pressure, membrane concentration, salting-out processing (for example, using ammonium sulfate or sodium sulfate etc.), Alternatively, it may be subjected to a fractional precipitation method using a hydrophilic organic solvent (eg, methanol, ethanol, acetone, etc.). By the above operation, the target protein of the present invention can be precipitated and purified.

  In the purification step, the purification step may be performed using heat treatment, isoelectric point treatment, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, reverse phase chromatography, or a combination thereof. .

  The purified enzyme containing the target protein obtained using the above method is preferably purified to such an extent that it shows a single band on electrophoresis (SDS-PAGE).

  The purified enzyme can be pulverized and distributed by, for example, freeze drying, vacuum drying, spray drying, or the like. Moreover, when using purified enzyme, it can be used in the state melt | dissolved in the buffer suitably according to the use. As the buffer solution, for example, a borate buffer solution, a phosphate buffer solution, a Tris-HCl buffer solution, and a GOOD buffer solution may be suitably selected according to the properties of the target protein and / or the experimental conditions or environment. . Furthermore, it can be stabilized by adding amino acids (for example, glutamic acid, glutamine, or lysine), serum albumin, and the like to the purified enzyme.

<4. Method for Measuring Glycated Protein According to the Present Invention>
The FAOD according to the present invention can be used in a method for measuring fructosyl amino acids. In addition, the fructosyl amino acid measurement method can be applied to the measurement of glycated proteins such as glycated hemoglobin. It should be noted that the substrate specificity of FAOD used for measurement of glycated hemoglobin is the reactivity with F-Lys in which the ε-amino group of the lysine residue in the blood protein is glycated. The α-amino group in a protein chain is one amino terminal amino acid residue, whereas there are usually many lysine residues that can be F-Lys in a protein chain. Therefore, the protein having FAOD activity in which the ratio of F-Lys reactivity to F-Val reactivity of the present invention is lower than that of the wild type is useful for measuring fructosyl amino acids.

  Hereinafter, a method for measuring a glycated protein using FAOD according to the present invention (hereinafter referred to as “the measuring method of the present invention”) will be described.

  The measurement method of the present invention is characterized in that the protein according to the present invention is allowed to act on at least a glycated amine. One embodiment of the measurement method of the present invention is shown below, but the present invention is not limited to this.

One embodiment of the measurement method according to the present invention is:
(1) A step of reacting a sample with a protease to decompose a glycated protein in the sample and preparing a glycated amine derived from the glycated protein in the sample (referred to as “first step” for convenience),
(2) a step of causing the protein according to the present invention to act on a glycated amine derived from a glycated protein in the sample obtained by the step (1) (referred to as “second step” for convenience), and
(3) A method for measuring a glycated protein comprising a step of measuring the amount of hydrogen peroxide generated or consumed oxygen in the step (2) (referred to as “third step” for convenience). .

(4-1) First Step The first step is a step of degrading a glycated protein in a sample to prepare a glycated amine derived from the glycated protein in the sample.

  The “glycated protein” means a protein in which a sugar is bound (glycated) to a part or all of amino acid residues constituting the protein. Although it does not specifically limit as glycated protein, For example, the thing (for example, HbA1c) etc. by which the alpha amino group of the amino terminal of protein was glycated are mentioned. The HbA1c exemplified above is used as an index for clinical diagnosis such as diabetes diagnosis.

  The protease is not particularly limited as long as it can decompose a glycated protein contained in a sample into a glycated amino acid or a glycated peptide. For example, proteases used in clinical tests can be preferably used.

  The “sample” is not particularly limited as long as it is a target to detect the presence or concentration of glycated protein. For example, in addition to whole blood, plasma, serum, blood cells, etc., urine, spinal fluid, etc. The present invention can also be applied to biological samples (i.e., samples collected from living organisms), drinking water such as juice, foods such as soy sauce and sauces. Since the method of the present invention can be applied to diabetes diagnosis, it is particularly useful for whole blood samples and blood cell samples. Although not particularly limited, when measuring glycated hemoglobin in erythrocytes, whole blood is lysed as it is, or erythrocytes separated from whole blood are lysed, and this hemolyzed sample is used as a sample for measurement. And it is sufficient.

  The specific conditions for reacting the sample with the protease are not particularly limited as long as the desired saccharified amine can be prepared, and may be appropriately determined depending on the concentration and type of the sample and the type and concentration of the protease. What is necessary is just to employ | adopt after considering suitable conditions.

  The concentration of protease that can be suitably used in the measurement method of the present invention is, for example, 0.1 U to 1 MU / ml (more preferably 1 U to 500 KU / ml, most preferably 5 U to 100 KU / ml). The concentration of the protease is not limited, and can be suitably determined by the experimenter or the user depending on the reaction conditions, the type and state of the sample, the technique of the experimenter, the type of reagent used, and the like.

  Examples of the “glycated amine” include glycated amino acids derived from glycated proteins contained in a sample.

(4-2) Second Step The second step is a step of allowing the protein according to the present invention to act on the glycated amine derived from the glycated protein in the sample obtained in the first step.

  The FAOD activity that the protein according to the present invention can have is not particularly limited, but the higher the FAOD activity, the more sensitive the glycated amine can be detected and the less the amount of FAOD protein used. Is preferable. The concentration of the FAOD protein that can be suitably used in the measurement method of the present invention is, for example, 0.1 to 500 U / ml (preferably 0.5 to 200 U / ml, most preferably 1.0 to 100 U / ml). is there. However, the concentration of the FAOD is not particularly limited, and can be appropriately determined according to the reaction conditions, the type and state of the sample, the experimenter's technique, the type of reagent used, and the like.

(4-3) Third Step The third step is a step of measuring the amount of hydrogen peroxide generated in the second step or the amount of consumed oxygen. As long as the third step is a method capable of measuring the amount of hydrogen peroxide or the amount of oxygen, the specific method is not particularly limited. Therefore, known methods can be applied as appropriate.

  Although the method used for the 3rd process is not limited, For example, an enzyme method etc. are mentioned. In the enzymatic method, a glycated protein in a sample is fragmented using an enzyme (for example, a protease) to the amino acid or peptide level. Next, FAOD is added to the resulting glycated amino acid and / or glycated peptide, and hydrogen peroxide is generated by an oxidation-reduction reaction. A POD and a reducing agent that develops color by oxidation are added to the sample, and an oxidation-reduction reaction is caused between the hydrogen peroxide and the reducing agent using the POD as a catalyst. The amount of hydrogen peroxide can be measured by coloring the reducing agent by the oxidation-reduction reaction and measuring the color intensity.

  POD suitable for the third step is derived from horseradish, microorganisms and the like. Moreover, the suitable use density | concentration of the said POD is 0.01-100 units / mL.

  A suitable method for measuring hydrogen peroxide in the third step is to use a coupler (4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazone (MBTH)) in the presence of POD. On the other hand, a Trinder reagent that produces a dye by oxidative condensation reaction of a phenolic, aniline, or toluidine chromogen, and a leuco dye that directly oxidizes and colors in the presence of POD can be used. These methods are known to those skilled in the art and can be readily used in general.

  Although it does not limit as said Trinder reagent, For example, phenol and its derivative (s) can be used.

  Examples of the coupler that can be suitably used in the third step include 4-aminoantipyrine, aminoantipyrine derivatives, vanillin diamine sulfonic acid, methylbenzthiazolinone hydrazone (MBTH), and sulfonated methylbenzthiazolinone hydrazone (SMBTH). Is mentioned.

  Although it does not specifically limit as said leuco pigment | dye which can be used conveniently in a 3rd process, For example, a triphenylmethane derivative, a phenothiazine derivative, a diphenylamine derivative etc. can be used.

  In the measurement of the amount of hydrogen peroxide in the third step, in addition to the color development method using the POD or the like, measurement methods using various sensor systems are generally known to those skilled in the art (for example, but not limited to, (See JP 2001-204494 A). Specifically, examples of electrodes used in various sensor systems include oxygen electrodes, carbon electrodes, gold electrodes, and platinum electrodes. In the present invention, these electrodes on which an enzyme is immobilized are used as a working electrode, and a buffer under conditions suitable for the present invention is provided together with a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / Cl electrode). A constant voltage is applied to the working electrode while it is inserted into the liquid and kept at a constant temperature, and a sample is added to measure the increase in current caused by hydrogen peroxide resulting from the enzyme reaction. .

  Further, as a method for measuring by an amperometric system using a carbon electrode, a gold electrode, a platinum electrode, or the like, there is a system using an immobilized electron mediator. For example, an enzyme and an electron mediator such as potassium ferricyanide, ferrocene, osmium derivatives, and phenazine methosulfate are adsorbed or immobilized on a polymer matrix by a covalent bond method as a working electrode, and a counter electrode (for example, a platinum electrode) Etc.) and a reference electrode (for example, an Ag / AgCl electrode) is inserted into a buffer solution under conditions suitable for the present invention and kept at a constant temperature. Subsequently, a constant voltage can be applied to the working electrode, and a sample can be added to measure the increase in current due to hydrogen peroxide resulting from the enzyme reaction.

  The amount of saccharified amine can also be measured by measuring the amount of oxygen consumed in the third step (for example, but not limited to, see JP-A-2001-204494). Specifically, an oxygen electrode is used, oxygen is immobilized on the electrode surface, inserted into a buffer solution under conditions suitable for the present invention, and kept at a constant temperature. A sample is added here, and the decrease value of the current is measured.

  When measuring by an amperometric system using a carbon electrode, a gold electrode, a platinum electrode, etc., these electrodes to which an enzyme is immobilized are used as a working electrode, a counter electrode (for example, a platinum electrode), and a reference electrode ( For example, an increase amount of current including a mediator is measured together with an Ag / AgCl electrode. As the mediator, potassium ferricyanide, ferrocene, osmium derivatives, phenazine methosulfate, and the like can be used.

  In the measurement method of the present invention, an inactive protein may be added to the system for performing each step in order to increase the stability of the protein of the present invention. Inactive proteins include serum albumins, globulins and fibrous proteins. A preferred protein is bovine serum albumin, with a preferred concentration in wt / vol being 0.05-1%. Preferred inactive proteins are those that do not contain protease impurities that cause enzymatic degradation.

  The measurement of fructosyl amino acid concentration is performed using a specific volume of the sample and a specific volume of the reagent. Absorbance measurement is performed as soon as possible after mixing and before significant absorbance changes due to fructosyl amino acids occur to measure the sample blank. A first absorbance measurement after 0.5-5 seconds is appropriate. The second absorbance measurement is typically 3-5 minutes at 37 ° C. at a fructosyl amino acid concentration of 1 mg / dL after the absorbance has become steady. Typically, the reagent is standardized with an aqueous or serum solution having a known fructosyl amino acid concentration.

<5. Glycated Protein Measurement Kit and Glycated Protein Sensor According to the Present Invention>
(5-1) Glycated hemoglobin measurement kit A glycated hemoglobin measurement kit according to the present invention (hereinafter referred to as “kit of the present invention”) is characterized by containing at least the protein according to the present invention. Although the form of the protein concerning this invention contained in the kit of this invention is not specifically limited, For example, forms, such as aqueous solution, suspension, or a lyophilized powder, can be employ | adopted. The lyophilized powder can be prepared according to a conventional method.

  The method for blending the additive is not particularly limited. For example, a method of adding an additive to a buffer containing a protein according to the present invention, a method of adding a protein according to the present invention to a buffer containing an additive, or a protein and a stabilizer according to the present invention in a buffer The method of mix | blending simultaneously is mentioned.

  The kit of the present invention may comprise a reagent composition comprising at least the protein according to the present invention, a peroxidase, and a chromogen.

  In the measurement method of the present invention, a peroxidase reaction is used to detect hydrogen peroxide derived from oxidation of fructosyl amino acids. Therefore, peroxidase and chromogen (hydrogen peroxide coloring reagent) are preferably used for the reagent composition. The peroxidase and chromogen (hydrogen peroxide coloring reagent) are not limited at all.

  The chromogen (hydrogen peroxide coloring reagent) used in the present invention is preferably stable in solution and low in bilirubin interference. Examples of the chromogen (hydrogen peroxide coloring reagent) that can be suitably used in the present invention include 4-aminoantipyrine or 3-methyl-2-benzothiazoline hydrazone (MBTH), and phenol or a derivative thereof or aniline or a derivative thereof. Examples thereof include a reagent comprising a combination of derivatives. The chromogen (hydrogen peroxide coloring reagent) used in the present invention may be benzidines, leuco dyes, 4-aminoantipyrine, phenols, naphthols and aniline derivatives.

  The peroxidase preferably used in the present invention is preferably a horseradish peroxidase. The peroxidase is commercially available with high purity and low cost. The enzyme concentration must be high enough for a rapid and complete reaction, preferably 1,000 to 50,000 U / L.

  In addition to the above configuration, the reagent composition may contain a buffer (for example, a borate buffer solution, a phosphate buffer solution, a Tris-HCl buffer solution, and a GOOD buffer solution). Furthermore, the reagent composition includes chelating reagents (such as EDTA and O-dianisidine) that capture ions that interfere with the enzyme reaction, ascorbate oxidase that eliminates ascorbic acid, which is a substance that interferes with the determination of hydrogen peroxide, Surfactants (such as Triton X-100 and NP-40) and various antibacterial and preservatives (such as streptomycin and sodium azide) may be included. These reagents may be a single reagent or a combination of two or more reagents.

  Although it does not specifically limit as said buffering agent, Arbitrary buffering agents which have sufficient buffering ability in the pH range of 6-8.5 can be used. Buffers in this pH range include phosphate, tris, bis-trispropane, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 2-morpholinoethanesulfonic acid monohydrate (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), 2- [4- (2-hydroxyethyl) -1 -Piperazinyl] ethanesulfonic acid (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid) (HEPES), and 3- [N-tris (hydroxymethyl) methylamino] -2-hydroxypropanesulfonic acid (TAPSO). Particularly preferred buffering agents are MES and PIPES. Moreover, especially a preferable concentration range is 20-200 mM, and is pH 6-7.

  The kit of the present invention includes, for example, FAOD, buffer solution, protease, POD, coloring reagent, chelating reagent that captures ions that interfere with the enzyme reaction, ascorbic acid that eliminates ascorbic acid that interferes with the determination of hydrogen peroxide. Includes oxidases, surfactants, stabilizers, excipients, antibacterial agents, preservatives, well plates, fluorescent scanners, automatic analyzers, and instruction manuals describing the measurement method according to the present invention on paper media It may be.

(5-2) Glycated protein sensor A glycated protein sensor according to the present invention (hereinafter referred to as “sensor of the present invention”) is a sensor used for detecting a glycated protein, and includes at least the protein according to the present invention. The sensor of the present invention is used for carrying out the measuring method of the present invention. Therefore, the sensor of this invention may be comprised by the article | item used for implementation of the measuring method of this invention. For the description of the article, the description of the buffering agent and the like in the section of the measurement method of the present invention and the kit of the present invention can be used.

  One embodiment of the sensor of the present invention can be used with the protein according to the present invention immobilized on a support. The support is not particularly limited as long as the protein according to the present invention can be immobilized, and a shape and material suitable for the properties of the protein may be used. The shape of the support is not particularly limited as long as it has a sufficient area to which the protein according to the present invention can be immobilized, and examples thereof include substrates, beads, and membranes. Examples of the support material include inorganic materials, natural polymers, and synthetic polymers.

  It should be noted that the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and obtained by appropriately combining technical means disclosed in different embodiments. Embodiments are also included in the technical scope of the present invention.

  Hereinafter, the present invention will be specifically described by way of examples. Table 1 shows the compositions of the activity measurement reagents used in the examples. The reagents used in the examples were those purchased from Nacalai Tesque unless otherwise specified.

  The FAOD activity measurement conditions in the examples are shown below.

-Activity measurement method-
The enzyme activity for each substrate was measured by the increase in the absorbance of the dye produced by the peroxidase reaction following the hydrogen peroxide produced by the enzyme reaction. After preheating the 3 ml of activity measuring reagent at 37 ° C. for 5 minutes, 0.1 ml of an enzyme solution previously diluted with an enzyme diluent (50 mM potassium phosphate buffer (pH 7.5)) is added to start the reaction. The reaction is carried out at 37 ° C. for 5 minutes, and the change in absorbance at 500 nm is measured (ΔOD _test / min). In the blind test, 0.1 ml of an enzyme diluent was added instead of the enzyme solution, and the change in absorbance was measured by performing the same operation as above (ΔOD _blank / min). The enzyme activity was calculated from the obtained absorbance change based on the following formula. The amount of enzyme that oxidizes 1 micromole of substrate per minute under the above conditions is defined as 1 unit (U).

-Calculation formula-
Activity value (U / ml) = {ΔOD / min (ΔOD _test −ΔOD _blank ) × 3.1 (ml) × dilution ratio} / {13 × 1.0 (cm) × 0.1 (ml)}
3.1 (ml): total liquid volume 13: mmol extinction coefficient 1.0 cm: cell optical path length 0.1 (ml): enzyme sample liquid volume <Reference Example 1. Cloning of FAOD gene>
The Aspergillus nidulans FAOD gene is cloned using the Aspergillus nidulans genome database (http://www.broad.mit.edu/annotation/genome/aspergillus#group/GenomesIndex.html) searched. A gene predicted to encode FAD-dependent oxidase was extracted from the genome database. Among the various genes obtained from the search results, Aspergillus nidulans FGSC A4 chromosome II ANcontig1.139 (http: // www. ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=29570992), the gene corresponding to 325868 to 327486 was cloned.

  Next, cDNA cloning of the FAOD gene was performed. Aspergillus nidulans A89 strain was added to GPY liquid medium (2% glucose, 0.5% casamino acid, 1% peptone, 0.5% yeast extract, 0.1% dipotassium monohydrogen phosphate, 0.05% Magnesium sulfate, 0.05% potassium chloride, 0.001% iron sulfate, pH 6.0) was cultured at 30 ° C. for 2 days. Total RNA was extracted from liquid-cultured Aspergillus nidulans A89 strain using ISOGEN (Nippon Gene). The total RNA was prepared according to the ISOGEN (Nippon Gene) protocol. MRNA was extracted from the prepared total RNA using Mag Extractor-mRNA (Toyobo Co., Ltd.). RT-PCR by RiverTra-Plus- (Toyobo Co., Ltd.) was performed using 0.1 μg of the obtained mRNA. In the first step reverse transcription reaction, an oligodeoxyribonucleotide P1 primer (5′-CTACATCTTTGCCTCATTCCTCCACCCCGG-3 ′) shown in SEQ ID NO: 19 was used. In the second-stage PCR, the P1 primer and the oligodeoxyribonucleotide P2 primer (5′-ATGACGCCCCGAGCCAACACCAAAATCATT-3 ′) shown in SEQ ID NO: 20 were used as primers. The obtained cDNA was blunt-ended with Blunting high (Toyobo Co., Ltd.) and subcloned into the SmaI site of the known plasmid pUC118. As a result of sequence analysis of the cDNA portion, the base sequence shown in SEQ ID NO: 2 was obtained. The cDNA coding region of the base sequence shown in SEQ ID NO: 2 is the 1314th base from the 1st base, the start codon is the 3rd base from the 1st base, and the stop codon TAG is the 1317th base from the 1315th base. It corresponds to the base of The amino acid sequence revealed from the base sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 1.

<Reference Example 2. Mass expression and purification of FAOD gene in E. coli, enzyme assay>
A large-scale expression in E. coli of a plasmid containing the FAOD gene having the base sequence shown in SEQ ID NO: 1 was attempted. A full-length cDNA region excluding the stop codon of the FAOD gene (up to the 1st to 1314th bases of the base sequence shown in SEQ ID NO: 2) was amplified by PCR. At that time, an NdeI cleavage site was inserted using the primer P3 (5′-GGAATTCCATATGACGCCCCGAGCCAACACCAAAATCATTGTC-3 ′) (“CATATG” in the above base sequence is an NdeI site) shown in SEQ ID NO: 21 on the N-terminal side of the amino acid sequence. An XhoI cleavage site was introduced using primer P4 (5′-CCGCTCGAGCATCTTTGCCTCATTCCTCCACCCCGGCAT-3 ′) (“CTCGAG” in the above base sequence is an XhoI site) shown in SEQ ID NO: 22 on the side. This DNA fragment was subcloned into the NdeI-XhoI site of the pET-23b vector (Novagen) so as to be in the forward direction with the T7 promoter. The prepared plasmid was named pIE201 as a FAOD expression plasmid.

  Escherichia coli BL21 CodonPlus (DE3) -RIL (Stratagene) was transformed with the plasmid pIE201. Then, transformant BL21 CodonPlus (DE3) -RIL (pIE201) showing ampicillin resistance was selected.

  As a medium for culturing the transformant, 200 ml of TB medium was dispensed into a 2 L Sakaguchi flask and autoclaved at 121 ° C. for 20 minutes. After allowing the medium to cool, ampicillin, which was separately sterile filtered, was added to the medium to a final concentration of 100 μg / ml. This medium was inoculated with 2 ml of BL21 CodonPlus (DE3) -RIL (pIE201) culture solution previously cultured in an LB medium containing 100 μg / ml ampicillin at 30 ° C. for 16 hours. Subsequently, aeration and agitation culture was performed at 30 ° C. for 24 hours. After completion of the culture, the cells were collected by centrifugation, suspended in 20 mM potassium phosphate buffer (pH 7.0), and the cells were sonicated. Next, the suspension was centrifuged to obtain a supernatant as a crude enzyme solution. The FAOD activity of the crude enzyme solution measured using a fructosyl valine-containing measurement reagent was about 4.0 U / ml.

  The obtained crude enzyme solution was subjected to denucleic acid removal with polyethyleneimine and ammonium sulfate fractionation, and dialyzed using 20 mM potassium phosphate buffer (pH 7.0). The sample after dialysis was purified using MagExtractor-His-tag- (manufactured by Toyobo) according to a prescribed protocol. Next, DEAE Sepharose CL-6B (manufactured by GE Healthcare Bioscience) is further separated and purified, followed by dialysis using 20 mM potassium phosphate buffer (pH 7.0), and purified enzyme preparation (IE201 ) The IE201 sample obtained by this method was confirmed to be single by SDS polyacrylamide gel electrophoresis.

  The enzyme activity of the IE201 preparation was measured using a measuring reagent containing F-Val and F-Lys, respectively, and the substrate specificity of the enzyme preparation was confirmed.

<Example 1. Production and purification of protein having fructosyl amino acid oxidase activity by functional modification of FAOD gene, enzyme assay>
Using the expression plasmid pIE201 and the synthetic oligonucleotides (SEQ ID NO: 11 and SEQ ID NO: 12), the mutation treatment operation was performed according to the prescribed protocol using the KOD-Plus Site-Directed Mutagenesis Kit (manufactured by Toyobo Co., Ltd.). The sequence was determined, and a recombinant plasmid (pIE201-N55G) encoding a FAOD mutant (SEQ ID NO: 3) in which the 55th asparagine of the amino acid sequence shown in SEQ ID NO: 1 was replaced with glycine was obtained. In the same manner, recombinant plasmids (pIE201-N55V, pIE201-N55S, pIE201-N55T) encoding a FAOD mutant in which the 55th asparagine of the amino acid sequence shown in SEQ ID NO: 1 was replaced with valine, serine, and threonine, respectively. ) Respectively.

  Further, mutation treatment was performed using the synthetic oligonucleotides shown in SEQ ID NO: 13 and SEQ ID NO: 14, the nucleotide sequence was determined, and the 268th phenylalanine of the amino acid sequence shown in SEQ ID NO: 1 was substituted with tryptophan. A recombinant plasmid (pIE201-F268W) having a polynucleotide encoding the FAOD mutant (SEQ ID NO: 5) was obtained. In the same manner, a set comprising a polynucleotide encoding a FAOD variant in which the 268th phenylalanine of the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine, serine, threonine, valine, leucine, methionine, cysteine, or histidine, respectively. Replacement plasmids (pIE201-F268A, pIE201-F268S, pIE201-F268T, pIE201-F268V, pIE201-F268L, pIE201-F268M, pIE201-F268C, pIE201-F268H) were obtained.

  Further, a mutation treatment operation was performed using the synthetic oligonucleotides shown in SEQ ID NO: 15 and SEQ ID NO: 16, the nucleotide sequence was determined, and the 286th phenylalanine of the amino acid sequence shown in SEQ ID NO: 1 was substituted with methionine. A recombinant plasmid (pIE201-F286M) having a polynucleotide encoding the FAOD mutant (SEQ ID NO: 7) was obtained. In the same manner, recombinant plasmids (pIE201-F286V, pIE201-F286L) having polynucleotides encoding FAOD mutants in which the 286th phenylalanine of the amino acid sequence shown in SEQ ID NO: 1 was replaced with valine and leucine, respectively. Acquired each.

  Further, a mutation treatment operation was performed using the synthetic oligonucleotides shown in SEQ ID NO: 17 and SEQ ID NO: 18, the nucleotide sequence was determined, and the 349th cysteine of the amino acid sequence shown in SEQ ID NO: 1 was substituted with valine A recombinant plasmid (pIE201-C349V) having a polynucleotide encoding the mutant (SEQ ID NO: 9) was obtained.

  After transforming BL21 CodonPlus (DE3) -RIL with each of the obtained plasmids, transformants showing ampicillin resistance were selected.

  In the same manner as in Reference Example 2, each transformant is cultured, and the resulting crude enzyme solution is purified to obtain each purified enzyme preparation (IE201-N55G, IE201-N55V, IE201-N55S, IE201-N55T). IE201-F268W, IE201-F268A, IE201-F268S, IE201-F268T, IE201-F268V, IE201-F268L, IE201-F268M, IE201-F268C, IE201-F268H, IE201-F286I, E201L28E -C349V). Each sample obtained by this method was confirmed to be single by SDS polyacrylamide gel electrophoresis.

<Example 2. Characterization of each FAOD protein>
Wild-type FAOD (IE201), and FAOD variants (IE201-N55G, IE201-N55V, IE201-N55S, IE201-N55T, IE201-F268W, IE201-F268A, IE201-F268S, IE201-F268T, IE201-F268V, IE201-F268V F268L, IE201-F268M, IE201-F268C, IE201-F268H, IE201-F286M, IE201-F286V, IE201-F286L, IE201-C349V) purified enzyme preparations, F-Val and F-Lys alone Table 2 shows the results of measurement using the measurement reagent contained in.

  In this example, the FAOD mutant is expressed as, for example, “IE201-N55S”, where “IE201” is a wild-type protein, “N” is a single letter of amino acid before substitution, and “55” is an amino acid of the wild-type protein The substitution position of the amino acid counted from the amino terminus in the sequence (SEQ ID NO: 1), “S”, indicates a single letter of the amino acid after substitution. As for other FAOD variants, the amino acid substitution positions and the amino acid residues before and after substitution can be read in the same manner as described above.

  As is clear from the results in Table 2, it was confirmed that all FAOD mutants had a reduced ratio of ε-fructosyl-L-lysine reactivity to fructosyl-L-valine reactivity relative to wild-type FAOD. It was done.

  In the measurement of specific activity, the amount of protein was measured by a colorimetric method based on the Bradford method.

  According to the present invention, it is possible to newly create and provide a fructosyl amino acid oxidase having substrate specificity useful for measuring fructosyl amino acids. In addition, it is possible to provide a method for measuring fructosyl amino acid using the fructosyl amino acid oxidase, a kit for measuring glycated protein, a glycated protein sensor, and the like. Therefore, it can contribute to the further spread of clinical tests based on preventive medicine.

Claims (14)

A protein characterized by the following (I) or (II):
(I) one or more amino acid residues of 55th asparagine, 268th phenylalanine, 286th phenylalanine, and 349th cysteine from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 are substituted; A protein having fructosyl amino acid oxidase activity and having a reduced ratio of ε-fructosyl-L-lysine reactivity to fructosyl-L-valine reactivity compared to a wild-type protein,
The 55th asparagine from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with one amino acid selected from the group consisting of glycine, valine, serine, and threonine;
The 268th phenylalanine from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with one amino acid selected from the group consisting of tryptophan, alanine, serine, threonine, valine, leucine, methionine, cysteine, and histidine And
286th phenylalanine from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with one amino acid selected from the group consisting of methionine, valine, and leucine,
A protein in which the cysteine at position 349 from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with valine;
(II) In the protein of (I) above , 10 amino acid residues other than the 55th, 268th, 286th and 349th amino acid residues from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 are substituted, Proteins that are deleted and / or added and have a reduced ratio of ε-fructosyl-L-lysine reactivity to fructosyl-L-valine reactivity relative to the wild-type protein. The protein according to claim 1, wherein the protein is any one of the following (a) to (e):
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
(B) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5;
(C) a protein consisting of the amino acid sequence shown in SEQ ID NO: 7;
(D) a protein consisting of the amino acid sequence shown in SEQ ID NO: 9;
(E) In the proteins (a) to (d) above , no more than 10 amino acids other than the 55th, 268th, 286th and 349th amino acid residues from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 A protein consisting of an amino acid sequence deleted, substituted and / or added.   The protein according to claim 1 or 2, wherein the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 is a fructosyl amino acid oxidase derived from Aspergillus nidulans.   The polynucleotide which codes the protein of any one of Claims 1-3.   The polynucleotide according to claim 4, which has the base sequence represented by SEQ ID NO: 4.   The polynucleotide according to claim 4, which has the base sequence represented by SEQ ID NO: 6.   The polynucleotide according to claim 4, which has the base sequence represented by SEQ ID NO: 8.   The polynucleotide according to claim 4, which has the base sequence represented by SEQ ID NO: 10.   A vector comprising the polynucleotide according to any one of claims 4 to 8.   A transformant transformed with the vector according to claim 9.   The manufacturing method of fructosyl amino acid oxidase including the process of culture | cultivating the transformant of Claim 10.   The measuring method of a fructosyl amino acid including the process of making the protein of any one of Claims 1-3 act on a saccharified amine.   A glycated protein measurement kit comprising at least the protein according to any one of claims 1 to 3.   A glycated protein sensor comprising at least the protein according to any one of claims 1 to 3.