Pentosidine measurement method and measurement kit

Abstract

Provided is a method for measuring pentosidine in a specimen, said method including: a step for decomposing the specimen with an amino acid degrading enzyme; a step in which the specimen after the decomposition step and a protein having activity for oxidatively decomposing pentosidine are brought into contact with each other; and a step for detecting changes caused by the contact, wherein the amino acid degrading enzyme and the protein having activity for oxidatively decomposing pentosidine are different from each other.

Description

[Technical field]

[0001] The present invention relates to a method for measuring pentosidine, and more particularly to a method for measuring pentosidine that reduces measurement errors and obtains accurate measurement values. [Background technology]

[0002] Pentosidine ((2S)-2-amino-6-[2-[[(4S)-4-amino-4-carboxybutyl]amino]imidazo[4,5-b]pyridin-4-yl]hexanoic acid) is a pentose with an equimolar crosslinked structure of lysine and arginine. It is known to accumulate in human skin in correlation with aging and the onset of diabetes, and to increase particularly in the onset of diabetes and end-stage nephropathy.

[0003] It is known that pentosidine can be quantified by HPLC using its fluorescence (Ex: 335 nm, Em: 385 nm) as an indicator after acid hydrolysis, and can also be quantified using immunochemical methods (e.g., ELISA) using monoclonal antibodies against pentosidine.

[0004] Pentosidine is known to be associated with schizophrenia in addition to aging and diabetes. For example, a method for testing schizophrenia has been disclosed that includes a step of measuring the amount of pentosidine in a biological sample (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5738346 Summary of the Invention [Problem to be solved by the invention]

[0006] Quantitative determination of pentosidine by immunochemical methods or instrumental analytical methods may be complicated and expensive. The present invention aims to provide a method for measuring pentosidine using a novel enzyme, which is cheaper and simpler than immunochemical methods or instrumental analytical methods, and in particular, to provide a method for measuring pentosidine that reduces measurement errors and obtains accurate measured values. [Means for solving the problem]

[0007] The present inventors discovered that a novel enzyme identified from a filamentous fungus is useful for quantifying pentosidine, and found that using this enzyme in combination with an amino acid-degrading enzyme can further reduce measurement errors, thereby completing the present invention.

[0008] The outline of the present invention is as follows. [1] A method for measuring pentosidine in a sample, comprising: Decomposing the sample with an amino acid decomposing enzyme; contacting the specimen after the decomposition step with a protein having an activity of oxidatively decomposing pentosidine; and detecting a change caused by said contact; wherein the amino acid decomposition enzyme and the protein having the activity of oxidatively decomposing pentosidine are different. [2] The measurement method according to [1], wherein a change in the amount of oxygen, hydrogen peroxide or ammonia is detected in the detection step. [3] The protein having the activity of oxidatively decomposing pentosidine has the following physicochemical properties: (1) Action: The activity of oxidatively decomposing pentosidine; and (2) Molecular weight by SDS-PAGE: 75,000-85,000 The measurement method according to [1] or [2], [4] The protein having the activity of oxidatively decomposing pentosidine is selected from the group consisting of the following (a) to (f): (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (b) a protein encoded by a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; (c) a protein consisting of an amino acid sequence having 75% or more identity to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (d) a protein encoded by a gene consisting of a nucleotide sequence having 75% or more identity to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; (e) a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 in which one or more amino acids have been deleted, substituted and / or added; or (f) a protein encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; The method for measuring the protein according to any one of [1] to [3], wherein the protein is any protein selected from the group consisting of: [5] The method according to any one of [1] to [4], wherein the protein having the activity of oxidatively decomposing pentosidine is derived from a filamentous fungus. [6] the protein having the activity of oxidatively decomposing pentosidine is pentosidine oxidase, The method according to any one of [1] to [5], wherein the amino acid degrading enzyme degrades amino acids contained in a sample, and the amino acids are selected from arginine, leucine, methionine, phenylalanine, tryptophan, and tyrosine. [6'] The method according to any one of [1] to [5], wherein the amino acid decomposition enzyme decomposes an amino acid selected from arginine, leucine, methionine, phenylalanine, tryptophan, and tyrosine. [7] The method according to any one of [1] to [6], wherein the amino acid decomposed by the amino acid decomposition enzyme is an amino acid in which a protein having an activity to oxidatively decompose pentosidine has a relative activity of 40% or more, when the activity of the protein having the activity to oxidatively decompose pentosidine toward pentosidine is taken as 100%. [8] The method according to any one of [1] to [7], wherein the amino acid decomposition enzyme is selected from the group consisting of amino acid oxidase, amino acid dehydrogenase, amino acid aminotransferase, amino acid decarboxylase, amino acid ammonia lyase, amino acid oxygenase, and amino acid hydrolase. [9] (i) an amino acid degrading enzyme; and (ii) a protein having an activity of oxidatively decomposing pentosidine A kit for measuring pentosidine in a sample, comprising:

[10] The protein having the activity of oxidatively decomposing pentosidine is selected from the group consisting of the following (a) to (f): (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (b) a protein encoded by a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; (c) a protein consisting of an amino acid sequence having 75% or more identity to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (d) a protein encoded by a gene consisting of a nucleotide sequence having 75% or more identity to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; (e) a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 in which one or more amino acids have been deleted, substituted and / or added; or (f) a protein encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; The kit according to [9], wherein the protein is any protein selected from the group consisting of:

[11] The kit according to [9] or

[10] , wherein the amino acid decomposition enzyme is an enzyme that decomposes an amino acid selected from arginine, leucine, methionine, phenylalanine, tryptophan, and tyrosine.

[12] A method for producing a reaction product of pentosidine derived from a sample, comprising: Decomposing the sample with an amino acid decomposing enzyme; a step of contacting the specimen after the decomposition step with a protein having an activity of oxidatively decomposing pentosidine wherein the amino acid degrading enzyme and the protein having the activity of oxidatively degrading pentosidine are different.

[13] The amino acid decomposition enzyme is Aplysia Escapin and / or Eastern Diamondback Rattlesnake ( Crotalus adamanteus The method for measuring amino acid oxidase according to any one of [1] to [8], wherein the amino acid oxidase is derived from

[14] The method according to any one of [1] to [8], wherein the amino acid degrading enzyme degrades amino acids contained in a sample, and the amino acids are selected from asparagine, glutamine, and histidine. [14'] The method according to any one of [1] to [7], wherein the amino acid decomposing enzyme decomposes an amino acid selected from asparagine, glutamine, and histidine.

[15] The kit according to any one of [9] to

[11] , further comprising (iii) at least one selected from a hydrogen peroxide detection reagent, an ammonia detection reagent, a pentosidine deamination product detection reagent, and an oxygen detection reagent. Effect of the Invention

[0009] According to the present invention, pentosidine can be detected and quantified simply and quickly by an enzymatic method, and in that case, measurement errors are reduced and accurate measurement values ​​can be obtained. [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows the results of a substrate concentration dependency test for the crude enzyme solution (Elution 1) fractionated from Sarocladium sp. using anion exchange chromatography. ΔOD (vertical axis) is plotted against the final pentosidine concentration (horizontal axis). Data from 20 minutes after the start of the reaction was used. [Diagram 2] 2 shows the results of a heat inactivation test of the crude enzyme solution (Elution 1). The results are from an analysis of the inactivation of enzyme activity by heat treatment, and correspond to data 20 minutes after the start of the reaction. [Diagram 3] 3 shows the results of measuring the concentration of hydrogen peroxide produced by the reaction of pentosidine with pentosidine oxidase. The concentration of hydrogen peroxide was measured by absorbance at 658 nm. [Figure 4] FIG. 4 shows the relationship between the final concentration of pentosidine and the increase in A658 (ΔA) caused by the oxidation of pentosidine. [Diagram 5] Figure 5 shows the estimated mechanism of the reaction in which pentosidine oxidase decomposes pentosidine. The figure shows that the amino groups of lysine and arginine, which constitute pentosidine, are oxidatively deaminated to produce hydrogen peroxide and ammonia. [Figure 6A] FIG. 6A shows the sequences of SEQ ID NO:1 and SEQ ID NO:2. [Figure 6B] FIG. 6B shows the sequences of SEQ ID NO:3 and SEQ ID NO:4. [Figure 6C] FIG. 6C shows the sequences of SEQ ID NO:5 and SEQ ID NO:6. [Figure 6D] FIG. 6D shows the sequences of SEQ ID NO:7 to SEQ ID NO:11. [Figure 6E] FIG. 6E shows the sequences of SEQ ID NO:12 to SEQ ID NO:14. [Figure 7] FIG. 7 shows the optimum pH range for PenOX2. [Figure 8] FIG. 8 shows the optimum temperature range of PenOX2. [Figure 9] FIG. 9 shows the range of thermostability of PenOX2. [Figure 10] FIG. 10 shows the stable pH range of PenOX2. [Figure 11] FIG. 11 shows the Km value of PenOX2 for pentosidine. [Figure 12] FIG. 12 shows the molecular weight of PenOX2. [Figure 13] FIG. 13 shows the substrate specificity of amino acid decomposition enzyme 1 measured in Example 13. [Figure 14] FIG. 14 shows the substrate specificity of amino acid decomposition enzyme 2 measured in Example 14. [Figure 15] FIG. 15 shows the substrate specificity of pentosidine oxidase measured in Example 15. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, details of a method for measuring pentosidine, which is one embodiment of the present invention (hereinafter also referred to as "the present embodiment"), will be described. However, the technical scope of the present invention is not limited only to the matters in this section, and the present invention can take various forms as long as it achieves its object.

[0012] (A protein that has the activity of oxidatively decomposing pentosidine) In one aspect, the present embodiment relates to a method for measuring pentosidine in a sample. As described above, pentosidine has a structure in which pentose and equimolar lysine and arginine are crosslinked. The "protein having the activity of oxidatively decomposing pentosidine" used in the measurement method of the present embodiment is not limited as long as it is a protein having such decomposition activity, and includes pentosidine oxidase having pentosidine oxidase activity and pentosidine dehydrogenase having pentosidine dehydrogenase activity.

[0013] The protein having the activity of oxidatively decomposing pentosidine, which will be described in the Examples below, is a novel enzyme, and has at least pentosidine oxidase activity. As used herein, "pentosidine oxidase activity" refers to the activity of oxidatively decomposing pentosidine, more specifically, the activity of oxidizing pentosidine to generate its deamination product, hydrogen peroxide, and ammonia, or the activity of consuming oxygen.

[0014] As used herein, "pentosidine dehydrogenase activity" refers to the activity of oxidatively decomposing pentosidine, more specifically, the activity of oxidizing pentosidine to generate its deamination product, reduced coenzyme, ammonia, or the activity of consuming oxidized coenzyme. Examples of the coenzyme referred to here include flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP). The reduced coenzyme may further reduce a mediator. The mediator is not particularly limited as long as it can donate and receive electrons between the coenzyme contained in the pentosidine dehydrogenase of the present invention. Examples of mediators include, but are not limited to, quinones, phenazines, ferricyanides, osmium salts or complexes, ruthenium salts or complexes, nitrosoanilines, aminoanilines, viologens, cytochromes, phenoxazines, phenothiazines, ferredoxins, ferrocenes, and derivatives thereof. Examples of quinones include naphthoquinone and its derivatives (e.g., naphthoquinone-4-sulfonic acid), phenanthrolinequinone and its derivatives, and phenanthrenequinone and its derivatives. Examples of phenazines include phenazine methosulfate (PMS) and its derivatives (e.g., 1-methoxy PMS, 1-ethoxy PMS). Examples of ferricyanides include potassium ferricyanide. Examples of osmium salts or complexes include osmium chloride and hexaammine osmium. Examples of ruthenium salts or complexes include ruthenium chloride and hexaammine ruthenium. Examples of nitrosoanilines include, but are not limited to, N,N-dimethyl-4-nitrosoaniline, N,N-bis-hydroxyethyl-4-nitrosoaniline, and derivatives thereof. Other mediators include those known to those skilled in the art. In this specification, unless otherwise specified, the term mediator does not include oxygen or hydrogen peroxide.

[0015] As long as it has the above-mentioned enzyme activity, any protein and a gene encoding the same are intended to be included in the scope of this embodiment without being limited to a specific sequence. Among proteins having the activity of oxidatively decomposing pentosidine, the nucleotide sequence and amino acid sequence of pentosidine oxidase from the genus Sarocladium ( Sarocladium The following will explain the enzyme derived from filamentous fungi of the genus Bacillus subtilis.

[0016] (Amino acid sequence of pentosidine oxidase) The amino acid sequence of pentosidine oxidase is not particularly limited as long as it has the above-mentioned enzyme activity. For example, an embodiment of the enzyme having the above-mentioned pentosidine oxidase activity includes a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Hereinafter, the proteins having the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 may be referred to as pentosidine oxidase 1 (or PenOX1) and pentosidine oxidase 2 (or PenOX2), respectively. The gene (g4462) encoding pentosidine oxidase 1 is expected to be composed of six exons and five introns, while the gene (g10122) encoding pentosidine oxidase 2 is expected to be composed of two exons and one intron.

[0017] Pentosidine oxidase having the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 is derived from the genus Sarocladium ( Sarocladium These enzymes are derived from filamentous fungi of the genus Bacillus subtilis. The base sequences of the genes encoding these enzymes are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. Figure 6 shows the amino acid sequences and base sequences of the enzymes.

[0018] The amino acid sequence of pentosidine oxidase may be an amino acid sequence having one or more amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, preferably several amino acids, deleted, substituted, added, etc., per unit, assuming that 100 amino acids in the amino acid sequence are one unit, in the amino acid sequence of a wild-type enzyme such as SEQ ID NO: 2 or SEQ ID NO: 4. Here, the range of "one to several" in "one to several amino acid deletions, substitutions, additions" of the amino acid sequence is not particularly limited, but preferably means about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably about 1, 2, 3, 4 or 5, per unit. Additionally, "amino acid deletion" means the absence or disappearance of an amino acid residue in a sequence, "amino acid substitution" means that an amino acid residue in a sequence is replaced with another amino acid residue, and "amino acid addition" means that a new amino acid residue is added, as if inserted, into the sequence.

[0019] Specific examples of "deletion, substitution, and addition of amino acids" include replacement of an amino acid with another chemically similar amino acid while maintaining pentosidine oxidase activity. For example, a hydrophobic amino acid may be replaced with another hydrophobic amino acid, or a polar amino acid may be replaced with another polar amino acid having the same charge. Such chemically similar amino acids are known in the art for each amino acid.

[0020] Specific examples of non-polar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged basic amino acids include arginine, histidine, and lysine. Furthermore, negatively charged acidic amino acids include aspartic acid and glutamic acid.

[0021] Further, the amino acid sequence of pentosidine oxidase may include amino acid sequences that have a certain level of sequence identity to the amino acid sequence of a wild-type enzyme such as SEQ ID NO: 2 or SEQ ID NO: 4, and for example, includes amino acid sequences that have 75% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and most preferably 95% or more identity to the amino acid sequence of pentosidine oxidase.

[0022] (The gene encoding pentosidine oxidase) The gene encoding pentosidine oxidase (hereinafter, sometimes referred to as "pentosidine oxidase gene") is not particularly limited as long as it contains a base sequence encoding the amino acid sequence of the enzyme having the above-mentioned pentosidine oxidase activity. In some embodiments, pentosidine oxidase is produced by expressing the pentosidine oxidase gene in the transformant.

[0023] As used herein, "gene expression" means that an enzyme encoded by a gene is produced in a manner that retains its inherent catalytic activity through transcription, translation, etc. "Gene expression" also includes high expression of a gene, i.e., production of an enzyme encoded by the gene in an amount exceeding the amount that would be naturally expressed by a host organism as a result of insertion of the gene.

[0024] The pentosidine oxidase gene may be a gene that, when introduced into a host organism, is capable of producing pentosidine oxidase via splicing after transcription of the gene, or may be a gene that is capable of producing pentosidine oxidase without splicing after transcription of the gene.

[0025] The pentosidine oxidase gene does not have to be completely identical to a gene (i.e., a wild-type gene) that is originally possessed by a source organism such as a filamentous fungus of the genus Sarocladium. As long as it is a gene that encodes an enzyme having the above-mentioned pentosidine oxidase activity, it may be DNA having a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence of the wild-type gene.

[0026] As used herein, the term "a base sequence that hybridizes under stringent conditions" refers to a DNA base sequence that can be obtained by using a DNA corresponding to a part of the base sequence of a wild-type gene, such as SEQ ID NO: 1 or SEQ ID NO: 3, as a probe and employing a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like.

[0027] As used herein, "stringent conditions" refer to conditions under which signals from specific hybrids are clearly distinguished from signals from non-specific hybrids, and vary depending on the hybridization system used and the type, sequence, and length of the probe. Such conditions can be determined by changing the hybridization temperature, washing temperature, and salt concentration.

[0028] For example, when even non-specific hybrid signals are strongly detected, the specificity can be increased by increasing the temperature for hybridization and washing and, if necessary, decreasing the salt concentration for washing. When even specific hybrid signals are not detected, the hybrids can be stabilized by decreasing the temperature for hybridization and washing and, if necessary, increasing the salt concentration for washing.

[0029] In some embodiments, specific examples of stringent conditions include the following. For example, a DNA probe is used as the probe, and hybridization is performed overnight (about 8 to 16 hours) using 5×SSC, 1.0% (w / v) blocking reagent for nucleic acid hybridization (manufactured by Boehringer Mannheim), 0.1% (w / v) N-lauroyl sarcosine, and 0.02% (w / v) SDS. Washing is performed twice for 15 minutes using 0.1 to 0.5×SSC and 0.1% (w / v) SDS, preferably 0.1×SSC and 0.1% (w / v) SDS. The temperature at which hybridization and washing are performed is 65° C. or higher, preferably 68° C. or higher.

[0030] Examples of DNA having a base sequence that hybridizes under stringent conditions include DNA obtained by hybridizing under the above-mentioned stringent conditions using a filter on which DNA having the base sequence of a wild-type gene derived from a colony or plaque or a fragment of the DNA is immobilized; and DNA that can be identified by hybridizing in the presence of 0.5 to 2.0 M NaCl at 40 to 75°C, preferably in the presence of 0.7 to 1.0 M NaCl at 65°C, and then washing the filter at 65°C using 0.1 to 1 x SSC solution (1 x SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Probe preparation and hybridization methods can be carried out in accordance with the methods described in Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997 (hereinafter, these documents are also referred to as "reference technical documents").

[0031] In addition, a person skilled in the art can appropriately set conditions for obtaining DNA having a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence of a wild-type gene, taking into account not only conditions such as the salt concentration of the buffer and temperature, but also other conditions such as probe concentration, probe length, and reaction time.

[0032] Examples of DNA containing a base sequence that hybridizes under stringent conditions include DNA that has a certain level of sequence identity to the base sequence of DNA having the base sequence of a wild-type gene used as a probe, and examples of such DNA include DNA that has a sequence identity of 75% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more to the base sequence of the wild-type gene.

[0033] An example of a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence of a wild-type gene is a base sequence having deletions, substitutions, additions, etc. of one to multiple, for example, 1 to 125, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 20, and preferably one to several, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, bases per unit of the base sequence of the wild-type gene, where 500 bases in the base sequence are considered to be one unit.

[0034] Here, "base deletion" means that a base is missing or lost in a sequence, "base substitution" means that a base in a sequence is replaced with another base, and "base addition" means that a new base is added, as if inserted.

[0035] An enzyme encoded by a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence of a wild-type gene is likely to be an enzyme having an amino acid sequence that has one or more, preferably several, amino acid deletions, substitutions, additions, etc. in the amino acid sequence of the enzyme encoded by the base sequence of the wild-type gene, but has the same enzymatic activity as the enzyme encoded by the base sequence of the wild-type gene.

[0036] Furthermore, the gene encoding the enzyme may contain a base sequence that is identical to or similar to the amino acid sequence of the enzyme encoded by the wild-type gene, but different from the wild-type gene, by utilizing the fact that there are several types of codons corresponding to one amino acid. Examples of base sequences in which codons have been modified from the base sequence of such a wild-type gene include the base sequence of SEQ ID NO: 5 (penox1) in which the codon of g4462 has been modified, and SEQ ID NO: 6 (penox2) in which the codon of g10122 has been modified (FIG. 6C). The base sequence in which codons have been modified is preferably, for example, a base sequence in which codons have been modified so as to be easily expressed in a host organism.

[0037] (Means for calculating sequence identity) The method for determining the sequence identity of a nucleotide sequence or an amino acid sequence is not particularly limited. For example, the sequence identity can be determined using a commonly known method by aligning a wild-type gene or an amino acid sequence of an enzyme encoded by the wild-type gene with a target nucleotide sequence or amino acid sequence, and using a program for calculating the degree of identity between the two sequences.

[0038] As a program for calculating the identity rate between two amino acid sequences or nucleotide sequences, for example, the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993) is known, and a BLAST program using this algorithm has been developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). In addition, Gapped BLAST, a program that determines sequence identity with higher sensitivity than BLAST, is also known (Nucleic Acids Res. 25: 3389-3402, 1997). Therefore, a person skilled in the art can use, for example, the above programs to search for sequences that show high sequence identity to a given sequence in a database. These are available, for example, at the internet website of the US National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0039] Although each of the above methods can be commonly used to search for sequences that show sequence identity in a database, the homology analysis of Genetyx Network version 12.0.1 (Genetyx) can also be used as a means for determining the sequence identity of individual sequences. This method is based on the Lipman-Pearson method (Science 227:1435-1441, 1985). When analyzing the sequence identity of a base sequence, a region that codes for a protein (CDS or ORF) is used if possible.

[0040] (Origin of the gene that codes for the enzyme) The gene encoding the enzyme is derived from a biological species capable of producing pentosidine oxidase. Examples of organisms from which the gene encoding the enzyme is derived include microorganisms such as filamentous fungi. Specific examples of microorganisms capable of producing pentosidine oxidase include the genus Sarocladium ( Sarocladium Examples include the genus.

[0041] As described above, the organism from which the enzyme-encoding gene is derived is not particularly limited, but it is preferable that the enzyme expressed in the transformant is not inactivated by the growth conditions of the host organism and exhibits activity. Therefore, the organism from which the enzyme-encoding gene is derived is preferably a microorganism whose growth conditions are similar to those of the host organism to be transformed by inserting the enzyme-encoding gene.

[0042] Among the physicochemical properties of enzymes having pentosidine oxidase activity, the following are some characteristic examples. Molecular weight by SDS-PAGE: 75,000-85,000 ·Optimal pH: pH approx. 6.5~8.0 The optimal pH is the pH at which the enzyme acts most suitably, and pentosidine oxidase can also act at a pH outside the above range. ·Optimal temperature: approx. 37~50℃ The optimum temperature is the temperature at which the enzyme acts most suitably, and pentosidine oxidase can also act at temperatures outside the above temperature range. Temperature stability: When stored at 30℃ for 10 minutes, pentosidine oxidase activity is maintained at 90% or more. When stored at 40℃ for 10 minutes, pentosidine oxidase activity is maintained at 50% or more. -pH stability: Pentosidine oxidase activity is maintained at 60% or more in the pH range of 4.0 to 9.0. Km value: The Km value for pentosidine is 1 mM or less. The Km value is the Michaelis constant, and the specific calculation method is not particularly limited, and the Km value can be calculated by freely selecting a known method. For example, the Km value can be calculated according to the Michaelis-Menten equation drawn by the Lineweaver-Burk plot method, as described in Example 9 below.

[0043] (Cloning of genes encoding enzymes by genetic engineering techniques) The gene encoding the enzyme can be inserted into various suitable known vectors. Furthermore, this vector can be introduced into a suitable known host organism to produce a transformant into which a recombinant vector (recombinant DNA) containing a gene encoding the enzyme has been introduced. Those skilled in the art can appropriately select a method for obtaining the gene encoding the enzyme, a method for obtaining information on the base sequence of the gene encoding the enzyme and the amino acid sequence of the enzyme, a method for producing various vectors, and a method for producing a transformant. In addition, as used herein, the terms transformation and transformant include transduction and transductants, respectively. A non-limiting example of cloning of a gene encoding an enzyme will be described below.

[0044] To clone a gene encoding an enzyme, a gene cloning method generally used can be used as appropriate. For example, chromosomal DNA or mRNA can be extracted from microorganisms or various cells capable of producing the enzyme by a conventional method, for example, the method described in the reference technical literature (see above). cDNA can be synthesized using the extracted mRNA as a template. A chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA obtained in this manner.

[0045] In some embodiments, the gene encoding the enzyme can be obtained by cloning using the chromosomal DNA or cDNA of the source organism having the gene as a template. The source organism of the gene encoding the enzyme is not particularly limited, but may be any of the above-mentioned Salicladium sp. Sarocladium For example, Salocladium sp. Sarocladium sp.) is cultured, water is removed from the resulting cells, and the cells are physically ground in a mortar or the like while being cooled in liquid nitrogen to obtain fine powder-like cell fragments, from which a chromosomal DNA fraction is extracted by a conventional method. For the chromosomal DNA extraction procedure, a commercially available chromosomal DNA extraction kit such as DNeasy Plant Mini Kit (Qiagen) can be used.

[0046] Next, the chromosomal DNA is used as a template to carry out a polymerase chain reaction (hereinafter referred to as "PCR") using primers complementary to the 5'-end sequence and the 3'-end sequence to amplify the DNA. There are no particular limitations on the primers as long as they are capable of amplifying a DNA fragment containing the gene. As an alternative method, DNA containing the target gene fragment can be amplified by an appropriate PCR such as the 5'RACE method or the 3'RACE method, and these can be linked to obtain DNA containing the full-length target gene.

[0047] Furthermore, the method for obtaining a gene encoding an enzyme is not particularly limited, and it is possible to construct a gene encoding an enzyme not by genetic engineering techniques but by, for example, chemical synthesis.

[0048] The base sequence of a PCR amplified product or a chemically synthesized gene can be confirmed, for example, as follows. First, the DNA whose sequence is to be confirmed is inserted into an appropriate vector in accordance with a conventional method to prepare a recombinant DNA. For cloning into a vector, commercially available kits such as TA Cloning Kit (Invitrogen); commercially available plasmid vector DNAs such as pUC19 (Takara Bio), pUC18 (Takara Bio), pBR322 (Takara Bio), pBluescript SK+ (Stratagene), and pYES2 / CT (Invitrogen); and commercially available bacteriophage vector DNAs such as λEMBL3 (Stratagene) can be used. In some embodiments, the recombinant DNA is used to transfect a host organism, for example, Escherichia coli ( Escherichia coli ), preferably E. coli JM109 strain (Takara Bio) or E. coli DH5α strain (Takara Bio) is transformed with the plasmid p53. The recombinant DNA contained in the obtained transformant may be purified using a QIAGEN Plasmid Mini Kit (Qiagen) or the like.

[0049] The base sequence of each gene inserted into the recombinant DNA can be determined by the dideoxy method (Methods in Enzymology, 101, 20-78, 1983) or the like. The sequence analyzer used for determining the base sequence is not particularly limited, but examples include Li-COR MODEL 4200L sequencer (Aloka), 370DNA sequence system (PerkinElmer), and CEQ2000XL DNA analysis system (Beckman). Then, based on the determined base sequence, the amino acid sequence of the protein to be translated, i.e., the enzyme, can be known.

[0050] (Construction of recombinant vectors containing genes encoding enzymes) A recombinant vector (recombinant DNA) containing a gene encoding an enzyme can be constructed by combining a PCR amplification product containing any of the genes encoding the enzyme with various vectors in a form that allows expression of the gene encoding the enzyme. For example, it can be constructed by excising a DNA fragment containing any of the genes encoding the enzyme with an appropriate restriction enzyme, and ligating the DNA fragment with a plasmid cleaved with an appropriate restriction enzyme. Alternatively, it can be obtained by ligating a DNA fragment containing the gene to which sequences homologous to the plasmid have been added at both ends with a DNA fragment derived from the plasmid amplified by inverse PCR using a commercially available recombinant vector construction kit such as In-Fusion HD Cloning Kit (manufactured by Clontech).

[0051] (Method of producing transformants) The method for producing a transformant is not particularly limited, and examples thereof include a method of inserting a gene encoding an enzyme into a host organism in a manner that allows the gene to be expressed according to a conventional method. In some embodiments, a DNA construct is produced in which any of the genes encoding an enzyme is inserted between an expression-inducing promoter and a terminator, and then a host organism is transformed with the DNA construct containing the gene encoding the enzyme, thereby obtaining a transformant that overexpresses the gene encoding the enzyme. In this specification, a DNA fragment consisting of an expression-inducing promoter-enzyme-encoding gene-terminator and a recombinant vector containing the DNA fragment, which are produced for transforming a host organism, are collectively referred to as a DNA construct.

[0052] The method for inserting the gene encoding the enzyme into a host organism in a manner that allows the gene to be expressed is not particularly limited, and examples of the method include a method of directly inserting the gene into a chromosome of the host organism using homologous recombination or non-homologous recombination; and a method of introducing the gene into the host organism by linking the gene onto a plasmid vector.

[0053] In the method using homologous recombination, a DNA construct can be ligated between sequences homologous to the upstream and downstream regions of the recombination site on a chromosome, and inserted into the genome of a host organism. In the method using non-homologous recombination, the homologous sequence can be inserted into the genome of a host organism even if it is not ligated to a DNA construct. The high expression promoter is not particularly limited, and examples thereof include the promoter region of the TEF1 gene (tef1), which is a translation elongation factor, the promoter region of the α-amylase gene (amy), the alkaline protease gene (alp) promoter region, and the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter region.

[0054] In the method using a vector, the DNA construct can be inserted into a plasmid vector used for transformation of a host organism in a conventional manner, and the corresponding host organism can be transformed in a conventional manner.

[0055] Such a suitable vector-host system is not particularly limited as long as it is a system that allows the enzyme to be produced in the host organism, and examples thereof include a system of pUC19 and a filamentous fungus, and a system of pSTA14 (Mol. Gen. Genet. 218, 99-104, 1989) and a filamentous fungus.

[0056] The DNA construct is preferably used by introducing it into the chromosome of the host organism. Alternatively, the DNA construct can be incorporated into an autonomously replicating vector (Ozeki et al. Biosci. Biotechnol. Biochem. 59, 1133 (1995)) for use without being introduced into the chromosome.

[0057] The DNA construct may contain a marker gene that allows the selection of transformed cells. The marker gene is not particularly limited, and examples thereof include genes that complement the auxotrophy of the host organism, such as pyrG, niaD, and adeA; and drug resistance genes against drugs, such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also preferably contains a promoter, terminator, and other control sequences (e.g., enhancer, polyadenylation sequence, etc.) that allow the overexpression of a gene encoding an enzyme in the host organism. The promoter is not particularly limited, and examples thereof include appropriate expression-inducing promoters and constitutive promoters, such as tef1 promoter, alp promoter, amy promoter, and gpd promoter. The terminator is also not particularly limited, and examples thereof include alp terminator, amy terminator, and tef1 terminator.

[0058] In the DNA construct, the expression control sequence of the gene encoding the enzyme is not necessarily required if the DNA fragment containing the gene encoding the enzyme to be inserted contains a sequence having an expression control function. In addition, when transformation is performed by co-transformation, the DNA construct may not need to have a marker gene.

[0059] A tag for purification can be added to the DNA construct. For example, by connecting an appropriate linker sequence upstream or downstream of the gene encoding the enzyme and connecting six or more base sequences encoding histidine, purification using a nickel column can be made possible.

[0060] The DNA construct may contain a homologous sequence necessary for marker recycling. For example, the pyrG marker can be removed on a medium containing 5-fluoroorotic acid (5FOA) by adding a sequence homologous to the sequence upstream of the insertion site (5' homologous recombination region) downstream of the pyrG marker, or by adding a sequence homologous to the sequence downstream of the insertion site (3' homologous recombination region) upstream of the pyrG marker. The length of the homologous sequence suitable for marker recycling is preferably 0.5 kb or more.

[0061] One embodiment of the DNA construct is, for example, a DNA construct in which the tef1 gene promoter, an enzyme-encoding gene, the alp gene terminator, and the pyrG marker gene are linked to an In-Fusion Cloning Site in the multiple cloning site of pUC19.

[0062] One embodiment of a DNA construct for inserting a gene by homologous recombination is a DNA construct in which a 5' homologous recombination sequence, a tef1 gene promoter, an enzyme-encoding gene, an alp gene terminator, a pyrG marker gene, and a 3' homologous recombination sequence are linked together.

[0063] One embodiment of a DNA construct for inserting a gene by homologous recombination and recycling a marker is a DNA construct linking a 5' homologous recombination sequence, a tef1 gene promoter, a gene encoding an enzyme, an alp gene terminator, a homologous sequence for marker recycling, a pyrG marker gene, and a 3' homologous recombination sequence.

[0064] When the host organism is a filamentous fungus, a method known to those skilled in the art can be appropriately selected as a method for transforming the filamentous fungus. For example, a protoplast PEG method using polyethylene glycol and calcium chloride after preparing a protoplast of the host organism can be used (see, for example, Mol. Gen. Genet. 218, 99-104, 1989 (supra); JP 2007-222055 A, etc.). A medium for regenerating the transformant is appropriate depending on the host organism and transformation marker gene used. For example, when Aspergillus oryzae ( A. oryzae ), Aspergillus sojae ( A.sojae When the pyrG gene is used as a transformation marker gene, the transformant can be regenerated, for example, in Czapek-Dox minimal medium (Difco) containing 0.5% agar and 1.2 M sorbitol.

[0065] Also, for example, in order to obtain a transformant, the promoter of the gene encoding the enzyme originally present on the chromosome of the host organism may be replaced with a high expression promoter such as tef1 using homologous recombination. In this case, it is also preferable to insert a transformation marker gene such as pyrG in addition to the high expression promoter. For example, for this purpose, a transformation cassette consisting of the upstream region of the gene encoding the enzyme--transformation marker gene--high expression promoter--all or part of the gene encoding the enzyme can be used with reference to the examples described in JP 2011-239681 A. In this case, the upstream region of the gene encoding the enzyme and all or part of the gene encoding the enzyme are used for homologous recombination.

[0066] The whole or a part of the gene encoding the enzyme can be used, including the region from the initiation codon to the middle of the gene. The length of the region suitable for homologous recombination is preferably 0.5 kb or more.

[0067] The production of a transformant can be confirmed by culturing the transformant under conditions in which the enzymatic activity of the enzyme is observed, and then detecting the target product in the culture obtained after culturing.

[0068] The production of a transformant may be confirmed by extracting chromosomal DNA from the transformant, performing PCR using the DNA as a template, and confirming that a PCR product that can be amplified is generated when transformation occurs. In this case, for example, PCR is performed using a combination of a forward primer for the nucleotide sequence of the promoter used and a reverse primer for the nucleotide sequence of the transformation marker gene, and it is confirmed that a product of the expected length is generated.

[0069] When transformation is carried out by homologous recombination, it is preferable to perform PCR using a combination of a forward primer located upstream of the upstream homologous region used and a reverse primer located downstream of the downstream homologous region used, and to confirm that a product of the expected length is produced when homologous recombination occurs.

[0070] (host organism) The host organism is not particularly limited as long as it is an organism that can produce the enzyme by transformation with a DNA construct containing a gene encoding the enzyme. Examples of the host organism include microorganisms and plants. Examples of the microorganism include Aspergillus ( Aspergillus ) microorganisms, Escherichia ( Escherichia ) microorganisms, Saccharomyces ( Saccharomyces ) microorganisms of the genus Pichia ( Pichia ) microorganisms, Schizosaccharomyces ( Schizosaccharomyces ) microorganisms, Zygosaccharomyces ( Zygosaccharomyces ) microorganisms, Trichoderma ( Trichoderma ) microorganisms, Penicillium ( Penicillium ) genus microorganism, Rhizopus ( Rhizopus ) microorganisms, Neurospora crassa ( Neurospora ) microorganisms of the genus Mucor ( Mucor ) genus microorganism, Acremonium ( Acremonium ) microorganisms, Fusarium ( Fusarium ) microorganisms, Neosartoria ( Neosartorya ) genus microorganisms, Byssochlamys ( Byssochlamys ) microorganisms, Talaromyces ( Talaromyces) microorganisms, Ageromyces ( Ajellomyces ) genus microorganism, Paracossidioides ( Paracoccidioides ) genus microorganisms, Ansinocarpus ( Uncinocarpus ) genus microorganisms, Coccidioides ( Coccidioides ) genus microorganisms, Alfroderma ( Arthroderma ) microorganisms, Trichophyton ( Trichophyton ) genus microorganisms, Exophylla ( Exophiala ) microorganisms, Capronia ( Capronia ) microorganisms, Cladophialophora ( Cladophialophora ) genus microorganisms, Macrohomina ( Macrophobia ) genus microorganisms, Leptosphaeria ( Leptosphaeria ) microorganisms of the genus Bipolaris ( Bipolaris ) genus microorganisms, Dochistroma ( Dothistroma ) microorganisms, Pyrenophora ( Pyrenophora ) microorganisms, Neofsicoccum ( Neofusicoccum ) genus microorganisms, Cetosphaeria ( Setosphaeria ) genus microorganisms, Baudoinia ( Baudoinia ) genus microorganism, Gaeumanomyces ( Gaeumannomyces ) genus microorganism, Marsonina ( Marssonina ) genus microorganisms, Sphaerulina ( Sphaerulina ) genus microorganisms, Sclerotinia ( Sclerotinia ) genus microorganisms, Magnaporce ( Magnaporthe ) genus microorganism, Verticillium ( Verticillium ) genus microorganism, Pseudocercospora ( Pseudocercospora ) genus microorganisms, Colletotrichum ( Colletotrichum ) genus microorganisms, Ophiostoma ( Ophiostoma ) genus microorganisms, Metalhisium ( Metarhizium ) genus microorganism, Sporothrix ( Sporothrix ) genus microorganisms, Sordaria ( Sordaria ) genus microorganisms, Arabidopsis ( Arabidopsis ) plants, with microorganisms and plants being preferred, provided that in all cases the host organism excludes humans.

[0071] Among filamentous fungi, taking into consideration safety and ease of cultivation, Aspergillus oryzae, Aspergillus sojae, and Aspergillus niger ( A. niger), Aspergillus tamarii ( A. tamarii ), Aspergillus awamori ( A. awamori ), Aspergillus usamii ( A. usami ), Aspergillus kawachii ( A.kawachii ), Aspergillus saitoi ( A. Saitoi ) and other Aspergillus microorganisms are preferred.

[0072] In this embodiment, the expression of a protein is not limited to the use of a host organism as described above. For example, an in vitro cell-free protein expression system can be suitably used, particularly when the purpose is not mass production such as commercial-scale production. The cell-free protein expression system has the advantage that it does not require cell culture and can easily purify the protein. In the cell-free protein expression system, a gene corresponding to the desired protein and a reaction solution containing the molecular mechanisms of transcription and translation, such as a cell lysate, are mainly used.

[0073] (Examples of genes that code for enzymes) Examples of genes encoding enzymes derived from the genus Sarocladium include genes g4462 and g10122 having the nucleotide sequences set forth in SEQ ID NOs: 1 and 3, respectively. The amino acid sequences of pentosidine oxidase 1 protein (PenOX1) and pentosidine oxidase 2 protein (PenOX2) are shown in SEQ ID NOs: 2 and 4, respectively.

[0074] The method for obtaining a gene encoding an enzyme from the genus Sarocladium and organisms other than the genus Sarocladium is not particularly limited, but for example, the gene can be obtained by performing a BLAST homology search on the genomic DNA of the target organism based on the base sequences of genes g4462 and g10122 (SEQ ID NO: 1 and SEQ ID NO: 3) to identify a gene having a base sequence with high sequence identity to the base sequences of genes g4462 and g10122. Alternatively, the gene can be obtained by identifying a protein having an amino acid sequence with high sequence identity to the amino acid sequences of pentosidine oxidase 1 and pentosidine oxidase 2 proteins (SEQ ID NO: 2 and SEQ ID NO: 4) based on the total protein of the target organism, and identifying a gene encoding the protein.

[0075] A gene encoding an enzyme obtained from the genus Sarocladium, or a gene encoding an enzyme having sequence identity to the enzyme, can be introduced into any host cell, such as an Aspergillus microorganism, as the host organism, to transform it.

[0076] (Transformant) One embodiment of the transformant is a transformant that has been transformed into a host organism such as a microorganism or a plant, into which one or a combination of genes has been inserted and which is then transformed so as to express the inserted genes.

[0077] Another embodiment of the transformant is a transformant into a host organism such as a microorganism or a plant, into which a gene (including promoter sequences other than the ORF) containing all or part of gene g4462 or g10122, and a DNA construct designed to express at a high or low level a transcription factor that controls the transcription of the gene, are inserted, and the transformant is transformed so as to express the inserted gene.

[0078] When the host organism is an organism that is recognized to be capable of producing pentosidine oxidase, such as the genus Sarocladium, it is desirable to constitutively express the inserted gene at a higher level than the endogenous expression, or to conditionally express the gene at the later stage of culture after cell proliferation. By culturing or growing such a transformant under conditions suitable for the host organism or the transformant due to the action of the transcription factor with a changed expression level, the host organism does not produce pentosidine oxidase, or if it does produce pentosidine oxidase, it can produce pentosidine oxidase at a detectable level or more.

[0079] Pentosidine oxidase can be produced by culturing the transformant under culture conditions suitable for the growth of the transformant using a medium suitable for the growth of the transformant. The culture method is not particularly limited, and examples thereof include solid culture and liquid culture methods performed under aeration or non-aeration conditions when the host organism is a filamentous fungus. The following mainly describes the production method when the host organism or wild-type organism is a filamentous fungus, but the present embodiment is not limited to the following description.

[0080] The medium may be any of ordinary media for culturing a host organism or a wild-type organism (hereinafter, these are collectively referred to as "host organism, etc."), that is, any synthetic medium or natural medium containing a carbon source, a nitrogen source, inorganic substances, and other nutrients in appropriate ratios. When the host organism, etc. is an Aspergillus microorganism, YMG medium or PPY medium described in the Examples below may be used, but is not particularly limited thereto.

[0081] The culture conditions for the transformant may be those of the host organism generally known to those skilled in the art. For example, when the host organism is a filamentous fungus, the initial pH of the medium is adjusted to 5 to 10, the culture temperature is 20 to 40° C., and the culture time is set appropriately to several hours to several days, preferably 1 to 7 days, more preferably 2 to 4 days. The culture means is not particularly limited, and aeration and stirring submerged culture, shaking culture, static culture, etc. can be used, but it is preferable to culture under conditions that provide sufficient dissolved oxygen. For example, examples of the medium and culture conditions for culturing an Aspergillus microorganism include shaking culture at 30° C. and 160 rpm for 3 to 5 days using YMG medium or PPY medium described in the Examples below.

[0082] The method for extracting pentosidine oxidase from the culture after the culture is completed is not particularly limited. For the extraction, the cells collected from the culture by filtration, centrifugation, or the like may be used as is, or the cells dried or further pulverized after collection may be used. The method for drying the cells is not particularly limited, and examples thereof include freeze drying, sun drying, hot air drying, vacuum drying, aeration drying, and reduced pressure drying.

[0083] Alternatively, instead of the above-mentioned treatment, the cells may be subjected to disruption treatment such as a method for disrupting the cells using a disrupting means such as an ultrasonic disrupter, French press, Dynomill, or mortar, a method for dissolving the cell walls of the cells using a cell wall-dissolving enzyme such as Yatalase, or a method for dissolving the cells using a surfactant such as SDS or Triton X-100. These methods may be used alone or in combination.

[0084] The target product can be purified from the obtained extract by purification processes such as centrifugation, filter filtration, ultrafiltration, gel filtration, separation based on solubility differences, solvent extraction, chromatography (adsorption chromatography, hydrophobic chromatography, cation exchange chromatography, anion exchange chromatography, reverse phase chromatography, etc.), crystallization, activated carbon treatment, membrane treatment, etc.

[0085] (substrate specificity) The protein having the activity of oxidatively decomposing pentosidine of the present embodiment has a high decomposition activity (substrate specificity) for pentosidine, and may have decomposition activity for other amino acids. For example, but not limited to, in one aspect, the protein having the activity of oxidatively decomposing pentosidine of the present embodiment has a relative activity of 40% or more, 50% or more, or 60% or more for one or more amino acids selected from arginine, leucine, methionine, phenylalanine, tryptophan and tyrosine, or all of these amino acids, when the activity for pentosidine is 100%. Furthermore, in one aspect, the protein having the activity of oxidatively decomposing pentosidine of the present embodiment has a relative activity of 10% or more, or 20% or more for one or more amino acids selected from asparagine, glutamine and histidine, or all of these, when the activity for pentosidine is 100%.

[0086] The relative activity and substrate specificity can be measured using techniques known to those skilled in the art using techniques and conditions that are the same as or similar to those used for the measurement of pentosidine. For example, the activity and substrate specificity can be measured using reaction rate with reference to the techniques described in the Examples below.

[0087] (Measurement method) The method for measuring pentosidine according to the present embodiment is as follows: Decomposing the sample with an amino acid decomposing enzyme; contacting the specimen after the decomposition step with a protein having an activity of oxidatively decomposing pentosidine; and detecting a change caused by said contact.

[0088] As used herein, the term "sample" refers to a subject, for example, a pentosidine solution in which pentosidine is to be quantified; liquid and solid components derived from a living body, such as blood, blood components (serum, plasma, blood cells, etc.), body fluids, and excrement. In one aspect, the sample is derived from a subject suffering from or suspected of suffering from a disease related to pentosidine. The sample is preferably blood or a blood component, and particularly preferably plasma. The sample does not necessarily contain pentosidine, and even if it does not contain pentosidine, the measurement method according to this embodiment can be used for analysis of the presence or absence of pentosidine (qualitative analysis). When the sample is derived from a living body, it can be derived from any living organism, such as a human, mouse, rat, or monkey. The sample collected from the living body may be used as is, or after any treatment.

[0089] (Amino acid decomposition enzyme) The measurement method of the present embodiment includes a step of decomposing a specimen with an amino acid decomposition enzyme. By performing the decomposition with the amino acid decomposition enzyme prior to contact with a protein having an activity of oxidatively decomposing pentosidine, measurement errors caused by the reaction of the protein having an activity of oxidatively decomposing pentosidine with other amino acids can be reduced, and more accurate measurement of pentosidine can be achieved.

[0090] The amino acid decomposition enzyme is an enzyme different from the protein having the activity of oxidatively decomposing the pentosidine, and is an enzyme that can preferentially decompose amino acids other than pentosidine. Examples of the amino acid decomposition enzyme include amino acid oxidase, amino acid dehydrogenase, amino acid aminotransferase, amino acid decarboxylase, amino acid ammonia lyase, amino acid oxygenase (hydrosylase), amino acid hydrolase, etc., and any one can be used in consideration of its substrate specificity. The amino acid decomposition enzyme can be used alone, or two or more kinds can be mixed or used in combination.

[0091] The amino acid decomposition enzyme is not particularly limited, and known enzymes can be used, and commercially available products may also be used. For example, a commercially available reagent such as an amino acid quantification kit can be used as the amino acid decomposition enzyme. The production method is also not limited, and for example, a gene encoding an amino acid decomposition enzyme (e.g., Escapin (SEQ ID NO: 15: Jumbo Sea Hare ( Aplysia California It is also possible to prepare a transformant by introducing a gene encoding the amino acid sequence of a mature peptide of Escapin derived from Escapin, and to obtain an amino acid decomposition enzyme from the medium in which the transformant is cultured.

[0092] As the amino acid decomposition enzyme, for example, those shown in the table below can be used alone or in combination, taking into consideration substrate specificity. [Table 1]

[0093] The amino acid decomposing enzyme is an enzyme capable of decomposing a desired amino acid under conditions that do not react with pentosidine. In one embodiment, when the activity of the amino acid decomposing enzyme against the most active (most decomposable) amino acid is taken as 100%, the relative activity against pentosidine under the same conditions is 30% or less, preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less.

[0094] The amino acid decomposition enzyme can be selected taking into consideration the types of amino acids likely to be contained in the sample to be measured and the substrate specificity of the protein having the activity of oxidatively decomposing pentosidine used in the measurement. In one aspect, when the activity of a protein having the activity of oxidatively decomposing pentosidine on pentosidine is taken as 100%, a protein having the activity of oxidatively decomposing pentosidine can be used that decomposes amino acids with a relative activity of 5% or more, 10% or more, 20% or more, 40% or more, or 60% or more.

[0095] In one embodiment, when the protein having the activity of oxidatively decomposing pentosidine is pentosidine oxidase (preferably, pentosidine oxidase having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or a modified version thereof, more preferably, pentosidine oxidase having the amino acid sequence of SEQ ID NO: 4 or a modified version thereof), an amino acid decomposition enzyme that decomposes one or more, two or more, three or more, four or more, or five or more amino acids selected from arginine, leucine, methionine, phenylalanine, tryptophan, and tyrosine, or preferably all of these amino acids, can be used. As such an amino acid decomposition enzyme, for example, the following amino acid decomposition enzymes can be used. An amino acid decomposition enzyme having a relative activity against pentosidine of 30% or less, preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less under the same conditions, when the activity against one or more amino acids selected from arginine, leucine, methionine, phenylalanine, tryptophan, and tyrosine is taken as 100%. A combination of amino acid degrading enzymes, in which, when the activity of the combined enzymes against any of the amino acids arginine, leucine, methionine, phenylalanine, tryptophan and tyrosine is taken as 100%, the relative activity against pentosidine under the same conditions is 30% or less, preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less. A combination of Escapin or a variant thereof showing similar substrate specificity and L-amino acid oxidase from Crotalus gracilis or a variant thereof showing similar substrate specificity. A combination of Escapin or a modified form thereof showing similar substrate specificity, and Eastern diamondback rattlesnake-derived L-amino acid oxidase or a modified form thereof showing similar substrate specificity, further comprising one or more, two or more, three or more, four or more, or all of the enzymes selected from histidine decarboxylase, asparaginase, aspartate decarboxylase, glutaminase, and glutamate decarboxylase.

[0096] In one aspect, when the protein having the activity of oxidatively decomposing pentosidine is pentosidine oxidase (preferably, pentosidine oxidase having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or a modified version thereof, more preferably, pentosidine oxidase having the amino acid sequence of SEQ ID NO: 4 or a modified version thereof), an amino acid decomposition enzyme that decomposes one or more amino acids selected from asparagine, glutamine and histidine in addition to arginine, leucine, methionine, phenylalanine, tryptophan and tyrosine, or all of these amino acids, can be used.

[0097] The conditions for decomposing a sample with an amino acid decomposition enzyme are not particularly limited as long as the desired amino acid can be decomposed and the amino acid decomposition enzyme does not react with pentosidine, and can be appropriately set depending on the amino acid decomposition enzyme used. For example, when amino acid oxidase is used as the amino acid decomposition enzyme, the amount can be appropriately selected depending on the amount of amino acid contained in the sample, reaction conditions, etc., and is 0.001 to 50 U / ml, preferably 0.01 to 10 U / ml. The pH can be adjusted to, for example, pH 3 to 12, preferably pH 4 to 11, taking into consideration the range in which the amino acid oxidase used acts. Any known pH adjuster and buffer can be used depending on the sample and the adjusted pH. The reaction temperature can be, for example, 15 to 65°C, preferably 20 to 60°C, taking into consideration the optimal temperature range of the amino acid oxidase used. The reaction time may be sufficient to decompose the desired amino acid, and the reaction can be performed for, for example, 1 to 120 minutes, preferably 2 to 60 minutes.

[0098] After the decomposition with the amino acid decomposition enzyme, the specimen after the decomposition step is contacted with a protein having the activity of oxidatively decomposing pentosidine, either directly or after undergoing steps such as heating, centrifugation, concentration, dilution, etc. as necessary.

[0099] The conditions for contacting a sample with a protein having the activity of oxidatively decomposing pentosidine are not particularly limited, as long as the conditions are such that pentosidine can be decomposed. For example, when pentosidine oxidase is used as a protein having an activity of oxidatively decomposing pentosidine, the amount is appropriately selected depending on the amount of pentosidine contained in the sample, reaction conditions, etc., and is 0.001 to 50 U / ml, preferably 0.01 to 10 U / ml. The pH can be adjusted to, for example, pH 4 to 10, preferably pH 5.5 to 9, taking into consideration the range in which the pentosidine oxidase used acts. As the pH adjuster and buffer, any known ones can be used depending on the sample and the adjusted pH. The reaction temperature can be, for example, 20 to 60°C, preferably 30 to 55°C, taking into consideration the optimal temperature range of the pentosidine oxidase used. The reaction time may be sufficient to decompose the desired amino acid, and the reaction can be performed for, for example, 1 to 120 minutes, preferably 2 to 60 minutes.

[0100] Then, a change caused by the contact is detected. As used herein, the term "change caused by the contact" refers to the presence or absence of a starting material such as pentosidine contained in a sample, or a reaction product or reaction product with a protein having an activity of oxidatively decomposing pentosidine, or a change over time in the amount thereof.

[0101] In a more specific embodiment, the method for measuring pentosidine is: (A) subjecting a specimen to the action of pentosidine oxidase in the presence of water and oxygen; and (B) measuring the amount of at least one of the reaction products or reaction products resulting from the action of pentosidine oxidase. may include.

[0102] Examples of the reaction products measured in the above step (B) include hydrogen peroxide, ammonia, and the deamination product of pentosidine. The amount of hydrogen peroxide, which is the reaction product, can be measured, for example, by a peroxidase reaction. The amount of ammonia, which is the reaction product, can be measured, for example, by the indophenol method, a method using Nessler's reagent, or a method of measuring the amount of NADH using an enzyme that uses ammonia as a substrate, such as glutamate dehydrogenase or NAD synthase. As used herein, the term "deamination product" refers to a product in which one or both of the amino groups of lysine and arginine constituting pentosidine are removed and replaced with oxygen, and at least one end is a keto acid. An example of such a deamination product is shown in FIG. 5. Examples of the reaction products measured in the above step (B) include oxygen. The amount of oxygen reduced by the enzyme reaction can be measured, for example, by an oxygen electrode. Alternatively, colorimetry can be performed by oxidizing manganese ions with oxygen based on the Winkler method.

[0103] It has been reported that the plasma pentosidine concentration is about 70% higher in schizophrenia patients (68.4 ng / mL) than in healthy subjects (mean ± SD 39.6 ± 7.8 ng / mL) (Arai et al., Journal of Psychiatry and Neurology (2012), Vol. 114, No. 2, pp. 101-107; Arai, M., et al. Arch Gen Psychiat, 67; 589-597, 2010). According to the measurement method of this embodiment, pentosidine is decomposed by an amino acid decomposition enzyme prior to contact with a protein having the activity of oxidatively decomposing pentosidine, thereby reducing the measurement error caused by the reaction of a protein having the activity of oxidatively decomposing pentosidine with other amino acids to less than 70%, preferably less than 60%, more preferably less than 50%, and enabling more accurate measurement of pentosidine, which is also very useful for diagnosing diseases related to pentosidine.

[0104] In another aspect, the present embodiment provides a kit for measuring pentosidine in a sample, comprising an amino acid decomposition enzyme and a protein having an activity of oxidatively decomposing pentosidine. The kit according to the present embodiment can be used to detect a reaction product or a reaction consumer between pentosidine and a protein having an activity of oxidatively decomposing pentosidine. The kit according to the present embodiment may further comprise at least one of a reaction buffer, a reaction product detection reagent, for example, a hydrogen peroxide detection reagent, an ammonia detection reagent, and a pentosidine deamination product detection reagent, or a reaction consumer detection reagent, for example, an oxygen detection reagent. The kit according to the present embodiment can also be used as an in vitro diagnostic agent, and can be suitably used, for example, for the diagnosis of diseases associated with pentosidine or a reaction product between pentosidine and pentosidine oxidase, such as diabetes and nephropathy.

[0105] Reagents for detecting hydrogen peroxide include 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenocyazine (DA-67) and N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine (DA-64), which can detect hydrogen peroxide with high sensitivity, as well as known color reagents such as Trinder's reagent. Reagents for detecting ammonia include a combination of phenol-sodium nitroprusside and an oxidizing agent such as sodium hypochlorite (indophenol method), Nessler's reagent, etc. Reagents for detecting oxygen include a combination of manganese ions, sodium hydroxide, and sulfuric acid.

[0106] Detection of reaction products using color reactions can be performed very simply and inexpensively compared to immunochemical methods or instrumental analytical methods. However, detection of reaction products or reaction products does not exclude other known quantitative or qualitative methods other than detection reagents, and may be appropriately adopted. For example, instead of detection reagents for hydrogen peroxide or ammonia, detection can be performed using devices such as enzyme sensors equipped with dedicated detection electrodes.

[0107] The above-mentioned method for detecting the reaction products or reaction products can also be used for detecting and even diagnosing diseases directly or indirectly associated with pentosidine or the respective reaction products or reaction products.

[0108] Furthermore, the present embodiment provides a method for producing a reaction product of pentosidine derived from a sample, comprising: Decomposing the sample with an amino acid decomposing enzyme; a step of contacting the specimen after the decomposition step with a protein having an activity of oxidatively decomposing pentosidine wherein the amino acid degrading enzyme and the protein having the activity of oxidatively degrading pentosidine are different. Each step can be carried out by referring to the description regarding the method for measuring pentosidine.

[0109] Hereinafter, the present embodiment will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various forms as long as the object of the present invention can be solved. EXAMPLES

[0110] Example 1: Cultivation of Sarocladium sp. and preparation of enzyme solution Culture medium used MEA medium: Malt extract agar (Oxoid) was dissolved in distilled water to a concentration of 50 g / L. YMG medium: Yeast extract 0.4%, Malt extract 1%, Glucose 0.4%, pH 5.5

[0111] Cultivation of strains Sarocladium sp. ( Sarocladium The F10012 strain was applied to MEA medium and cultured at 24°C for 7 to 10 days until a sufficient amount of mycelium was obtained. The obtained mycelium was inoculated into 250 mL of YMG medium in a 1 L flask and cultured with shaking at 30°C for 3 days.

[0112] Preparation of crude enzyme solution The YMG medium in which the bacteria were cultured was filtered using Miracloth (Merck Millipore) to remove the bacteria and obtain the culture supernatant. The culture supernatant was concentrated using an ultrafiltration membrane (Vivaspin 20-3k, GE Healthcare) and diluted with 50 mM potassium phosphate buffer (pH 7.5) several times to remove low molecular weight compounds, and the YMG medium was replaced with potassium phosphate buffer.

[0113] - Crude purification of the target enzyme The buffer-exchanged crude enzyme solution was fractionated using an ion exchange chromatography column (HiTrap Q Sepharose Fast Flow 1 mL, GE Healthcare). The specific procedure is as follows.

[0114] First, the crude enzyme solution was loaded onto a column equilibrated with 50 mM potassium phosphate buffer (pH 7.5) to allow the enzyme to be adsorbed onto the column. After that, the column was washed with 5 mL of potassium phosphate buffer to elute unadsorbed proteins.

[0115] Thereafter, 5 mL of each of potassium phosphate buffer containing 0.25 M, 0.5 M, 0.75 M, and 1.0 M sodium chloride was passed through the column in sequence to elute the proteins adsorbed to the column.

[0116] The liquid eluted from the column when the crude enzyme solution was loaded was called "Flow through," the liquid eluted during washing with buffer was called "Start buffer," and the liquid eluted with the buffer containing sodium chloride was called "Elution 1," "Elution 2," "Elution 3," and "Elution 4," respectively, and each was collected in a different container.

[0117] (Example 2) Method for measuring pentosidine oxidase activity - Activity measurement of crude enzyme solution The activity was measured using the liquid eluted from the ion exchange chromatography column as a sample. 50 μL of the sample was mixed with 25 μL of 4 mM pentosidine (free form) (Peptide Institute, 3-trifluoroacetate (TFA) salt) dissolved in 100 mM potassium phosphate buffer (pH 8.0) and 25 μL of oxidase coloring reagent (4 U / mL peroxidase (TOYOBO), 1.8 mM 4-aminoantipyrine (Fluka), 2 mM TOOS (Dojindo)) and reacted at room temperature.

[0118] A 96-well microwell plate (Nunc) was used for the reaction. A blank was prepared by adding 100 mM potassium phosphate buffer (pH 8.0) instead of the substrate solution. The absorbance of the reaction solution and the blank solution was measured at 555 nm, and the strength of the enzyme activity was evaluated based on the difference in absorbance (ΔOD).

[0119] ·Substrate concentration dependence test The pentosidine oxidase activity of the crude enzyme solution was measured using various concentrations of substrate to evaluate the change in activity with respect to the substrate concentration. The concentrations of the substrate solutions used were 0.13 mM, 0.25 mM, 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM.

[0120] Thermal deactivation test The crude enzyme solution was heat-treated at 80° C. for 1 hour to denature the protein. The pentosidine oxidase activity of this heat-treated sample was measured according to the activity measurement method described above and compared with that of an unheated sample.

[0121] (Example 3) Pentosidine oxidase activity analysis of Sarocladium sp. enzyme solution Sarocladium sp. ( SarocladiumThe reactivity of the culture supernatant of E. sp. fractionated on an ion exchange chromatography column with pentosidine was analyzed. As a result, strong activity was observed in Elution 1, which was eluted with potassium phosphate buffer containing 0.25 M sodium chloride, suggesting that it contains pentosidine oxidase. Substrate concentration dependence test (Figure 1) and heat inactivation test (Figure 2) were performed on Elution 1, and it was found that the enzyme activity increased in a substrate concentration-dependent manner and was completely inactivated by heat treatment. This indicates that the pentosidine oxidase activity observed in Elution 1 is derived from the enzyme.

[0122] Example 4: Sequencing of pentosidine oxidase from Sarocladium sp. The above results and the results of Sarocladium sp. Sarocladium Based on the complete genome sequence information of P. sp., two genes (SEQ ID NO: 1 and SEQ ID NO: 3) and their amino acid sequences (SEQ ID NO: 2 and SEQ ID NO: 4) that are presumed to be pentosidine oxidase were identified.

[0123] Example 5: Heterologous recombinant expression of pentosidine oxidase from Sarocladium sp. in Aspergillus sojae To analyze the enzyme activities of the two pentosidine oxidases identified above, heterologous recombinant expression was performed using the koji mold Aspergillus sojae as a host.

[0124] -Preparation of expression vector The base sequences of SEQ ID NOs: 5 and 6, which were codon-modified for expression in Aspergillus oryzae based on the amino acid sequences of SEQ ID NOs: 2 and 4, were obtained by artificial gene synthesis, respectively.

[0125] As the expression cassette for expressing the pentosidine oxidase genes (penox1, penox2) of SEQ ID NO:5 and SEQ ID NO:6, Ptef (748 bp upstream of the tef1 gene, SEQ ID NO:7), which is the promoter sequence of the translation elongation factor gene tef1, was used as the promoter, and Talp (800 bp downstream of the alp gene, SEQ ID NO:8), which is the terminator sequence of the alkaline protease gene alp, was used as the terminator.

[0126] The selection marker used was the pyrG3 gene (1,487 bp including 56 bp upstream, 896 bp coding region, and 535 bp downstream, SEQ ID NO: 9), which complements the uracil / uridine requirement and enables the introduction of multiple copies of the gene (see JP 2018-068292 A). These Ptef, Talp, and pyrG3 genes were derived from the Aspergillus sojae ( Aspergillus sojae The gene was obtained by PCR using the genomic DNA of the NBRC4239 strain as a template.

[0127] Next, an In-Fusion HD Cloning Kit (Clontech) was used to link each DNA. For example, when linking Ptef to the penox1 gene and Talp, a DNA fragment was amplified by PCR using a reverse primer of SEQ ID NO: 10 for Ptef and a forward primer of SEQ ID NO: 11 for Talp. At this time, the reverse primer for amplifying Ptef of SEQ ID NO: 10 has a 15-bp sequence complementary to the 5'-end of the penox1 gene (SEQ ID NO: 5) added to its 5'-end, and the forward primer for amplifying Talp of SEQ ID NO: 11 has a 15-bp sequence homologous to the 3'-end of the penox1 gene (SEQ ID NO: 5) added to its 5'-end, so that Ptef, penox1 gene and Talp can be linked by the infusion reaction. In this manner, expression vectors p19-pG3-penox1 and p19-pG3-penox2 were prepared in which Ptef, the penox1 gene or the penox2 gene, Talp and pyrG3 were linked in that order, Ptef-penox1-Talp-pyrG3 and Ptef-penox2-Talp-pyrG3, respectively, were inserted into the multicloning site of the pUC19 plasmid.

[0128] - Creation and cultivation of Aspergillus oryzae expression strains Using the transformation plasmids p19-pG3-penox1 and p19-pG3-penox2 obtained above, we transformed an Aspergillus sojae pyrG gene disruptant (a strain lacking 48 bp upstream, 896 bp coding region, and 240 bp downstream of the pyrG gene) by the protoplast PEG method, and obtained 9 As-penox1 and 6 As-penox2 transformed strains in which multiple copies of the penox1 and penox2 expression cassettes were inserted.

[0129] The obtained Aspergillus sojae transformants, As-penox1 and As-penox2, were inoculated into 15 mL of PPY liquid medium (2% (w / v) pinedicle, 1% (w / v) polypeptone, 0.5% (w / v) yeast extract, 0.5% (w / v) potassium dihydrogen phosphate, 0.05% (w / v) magnesium sulfate heptahydrate) in a 50 mL Erlenmeyer flask and cultured with shaking at 30°C for 4 to 5 days.

[0130] Preparation of mycelial extract The culture solution of each of the As-penox1 and As-penox2 strains was filtered using Miracloth (Merck Millipore), and the culture supernatant was removed to obtain the bacterial cells. After resuspending in 15 mL of 10 mM potassium phosphate buffer (pH 7.5), the bacterial cells were disrupted using Micro Smash MS-100R (Tomy Seiko). The disrupted bacterial cell solution was centrifuged at 15,000 rpm for 15 minutes, and the supernatant was collected as a crude enzyme solution.

[0131] Measurement of L-arginine oxidation activity in mycelial extracts 200 μL of each crude enzyme solution was mixed with 380 μL of 7.1 U / mL peroxidase, 0.70 mM 4-aminoantipyrine, and 0.79 mM TOOS solution dissolved in 150 mM potassium phosphate buffer (pH 7.0) and incubated at 37°C for 5 minutes, after which 20 μL of 60 mM L-arginine solution was added, stirred, and reacted at 37°C for 5 minutes. 555The time course of change in was measured using a spectrophotometer (U-3900, Hitachi High-Tech Science Corporation). A control experiment was performed by adding 20 μL of ion-exchanged water instead of 20 μL of 60 mM L-arginine solution. The amount of enzyme that produces 1 μmol of hydrogen peroxide per minute at 37°C was defined as 1 unit (U) and calculated according to the following formula.

[0132] Activity (U / mL)={(ΔAs-ΔA0)×0.6×df}÷(39.2×0.5×0.2) ΔAs: A of the reaction solution per minute 555 Amount of change ΔA0: A per minute in the control experiment 555 Amount of change 39.2: Millimolar extinction coefficient of the quinone imine dye produced by the reaction (mM -1 ·cm -1 ) 0.5: Molar number of quinoneimine dyes produced by 1 mol of hydrogen peroxide 0.6: Total volume of reaction solution (mL) df: dilution factor 0.2: Volume of enzyme solution (mL)

[0133] The maximum L-arginine oxidation activities of the crude enzyme solutions of As-penox1 and As-penox2 strains were 0.009 U / mL (As-penox1-15 strain) and 5.1 U / mL (As-penox2-16 strain), respectively.

[0134] (Example 6) Purification of mycelium-extracted recombinant penox2 The crude enzyme solution of the As-penox2-16 strain was fractionated using an anion exchange chromatography column (HiScreen CaptoQ, GE Healthcare) after buffer replacement with 10 mM potassium phosphate buffer (pH 7.5). First, the crude enzyme solution was loaded onto a column equilibrated with 10 mM potassium phosphate buffer (pH 7.5), and the enzyme was adsorbed onto the column. The column was then washed with 10 mM potassium phosphate buffer (pH 7.5) to elute unadsorbed proteins. The sodium chloride concentration in the 10 mM potassium phosphate buffer (pH 7.5) was then linearly increased from 0 mM to 40 mM to elute the proteins adsorbed onto the column. Fractions showing L-arginine oxidation activity were analyzed by SDS-PAGE, and fractions not containing contaminating proteins were collected as purified PenOX2. The recovered purified PenOX2 solution was concentrated using an ultrafiltration membrane (Amicon Ultra 15-30k, Merck) until the L-arginine oxidation activity reached 24 U / mL, and was used in a pentosidine quantification test.

[0135] (Example 7) Pentosidine Quantitative Test The following reagents were prepared, and pentosidine was measured using Bio Majesty JCA-BM1650 (manufactured by JEOL Ltd.). (Sample: Pentosidine solution) 0.2 μM, 0.4 μM, 0.6 μM, 1.0 μM, 2.0 μM or 4.0 μM pentosidine solution (prepared using the same pentosidine as in Example 2)

[0136] (First reagent: Leuco dye, peroxidase solution) 120mM potassium phosphate buffer (pH 7.0) 0.2 mM DA-67 (10-(Carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, sodium salt) (Wako Pure Chemical Industries, Ltd.) 3.0U / mL peroxidase

[0137] (Second reagent: PenOX2 solution) 120mM potassium phosphate buffer (pH 7.0) 24U / mL PenOX2

[0138] 25 μL of the sample was added to 50 μL of the first reagent and incubated at 37° C. for 5 minutes, after which 25 μL of the second reagent was added and the pentosidine oxidation reaction by PenOX2 and the detection reaction of hydrogen peroxide produced by the reaction were allowed to proceed at 37° C. for 5 minutes.

[0139] In the hydrogen peroxide detection reaction, peroxidase is consumed and DA-67 is oxidized to methylene blue, which changes color and increases the absorbance (A 658 As an example, the absorbance (A 658 The relationship between the amount of PenOX2 and the amount of the second reagent is shown in Figure 3. 658 An increase in was confirmed.

[0140] Subsequently, A caused by the oxidation of pentosidine 658 The increase (ΔA) was calculated according to the following formula: ΔA = (absorbance 5 minutes after adding the second reagent) - (absorbance just before adding the second reagent x 0.75) (Since the concentration of the composition in the reaction solution becomes 0.75 times (75 / 100 times) by adding the second reagent, The absorbance immediately before the addition of the second reagent was multiplied by 0.75 and considered to be the absorbance immediately after the addition of the second reagent.

[0141] A correlation was established between the final pentosidine concentration and ΔA (Figure 4). Therefore, it was shown that PenOX2 exhibits pentosidine oxidation activity and can be used to quantify pentosidine. Although the results are not shown, PenOX1 also exhibited pentosidine oxidation activity.

[0142] (Example 8) Purification of recombinant penox2 secreted by hyphae The mycelium culture solution of As-penox2 strain was filtered using Miracloth (Merck Millipore) to recover the mycelium culture supernatant. 75 mL of the obtained mycelium culture supernatant was filtered through a syringe filter with a pore size of 0.2 μm, and then concentrated with an ultrafiltration membrane (Amicon Ultra 15-30k, Merck). Ammonium sulfate was gradually added to the concentrated solution to 70% saturation, and the solution was left at 4° C. for 2 hours, and then centrifuged (15,000 rpm, 4° C., 5 minutes) to precipitate excess protein, and the supernatant was recovered. The recovered supernatant was concentrated with an ultrafiltration membrane (Amicon Ultra 0.5-30k, Merck).

[0143] After adding 50 mM potassium phosphate buffer (pH 7.5) containing 2 M ammonium sulfate, the mixture was fractionated using a hydrophobic interaction chromatography column (HiTrap Butyl Fast Flow 1 mL, manufactured by GE Healthcare). The specific procedure is as follows.

[0144] First, the crude enzyme solution was loaded onto a column equilibrated with 50 mM potassium phosphate buffer (pH 7.5) containing 2 M ammonium sulfate. After the enzyme was adsorbed onto the column, the column was washed with 10 mL of 50 mM potassium phosphate buffer (pH 7.5) containing 2 M ammonium sulfate to elute unadsorbed proteins.

[0145] The proteins adsorbed to the column were then eluted by sequentially passing 5 mL each of 50 mM potassium phosphate buffer (pH 7.5) containing 1.5 M, 1.3 M, and 1.15 M ammonium sulfate, followed by 10 mL of 50 mM potassium phosphate buffer (pH 7.5) containing 1 M ammonium sulfate, and 5 mL of 50 mM potassium phosphate buffer (pH 7.5) without ammonium sulfate through the column.

[0146] The liquid eluted from the column when the crude enzyme solution was loaded was named "Flow through 1." The liquid eluted during washing with a buffer containing 2 M ammonium sulfate was named "Elution 1." The liquids eluted with buffers containing 1.5 M, 1.3 M, 1.15 M, and 1 M ammonium sulfate were named "Elution 2," "Elution 3," "Elution 4," and "Elution 5," respectively. The liquid eluted with a buffer containing no ammonium sulfate was named "Elution 6." Each was collected in a different container.

[0147] The fractionated samples were analyzed for reactivity with pentosidine. As a result, strong activity was observed in Elution 5 eluted with potassium phosphate buffer containing 1M ammonium sulfate, suggesting that it contains pentosidine oxidase (PenOX2). This Elution 5 was concentrated using an ultrafiltration membrane (Amicon Ultra 15-30k, Merck), and the buffer was replaced with 50 mM potassium phosphate buffer (pH 7.5) that does not contain ammonium sulfate, and then concentrated again using an ultrafiltration membrane (Amicon Ultra 15-30k, Merck). This was fractionated using an ion exchange chromatography column (HiTrap Q Sepharose Fast Flow 1mL, GE Healthcare). The specific procedure is as follows.

[0148] First, the crude enzyme solution was loaded onto a column equilibrated with 50 mM potassium phosphate buffer (pH 7.5) to allow the enzyme to be adsorbed onto the column. After that, the column was washed with 5 mL of 50 mM potassium phosphate buffer (pH 7.5) to elute unadsorbed proteins.

[0149] The protein adsorbed to the column was then eluted by passing 1 mL of 0.1 M sodium chloride in 50 mM potassium phosphate buffer (pH 7.5) five times, 1 mL of 0.175 M sodium chloride in the same buffer five times, and 5 mL of 1 M sodium chloride in the same buffer once.

[0150] The liquid eluted from the column when the crude enzyme solution was loaded was named "Flow through 2", the liquid eluted during washing with buffer was named "Elution 7", and the liquids eluted with a buffer containing sodium chloride were named "Elution 8-1", "Elution 8-2", "Elution 8-3", "Elution 8-4", "Elution 8-5", "Elution 9-1", "Elution 9-2", "Elution 9-3", "Elution 9-4", "Elution 9-5", and "Elution 10", respectively, and were collected in different containers.

[0151] The fractionated samples were analyzed for reactivity with pentosidine. As a result, strong activity was observed in Elution 9-1 and Elution 9-2 eluted with potassium phosphate buffer containing 0.175M sodium chloride, suggesting that pentosidine oxidase was contained. When an equal amount of the active fractions Elution 9-1 and Elution 9-2 were mixed and analyzed by SDS-PAGE, a nearly single band was obtained (molecular weight approximately 80,000). The obtained active fraction was used to determine the following physicochemical properties.

[0152] (Example 9) Physicochemical properties of PenOX2 produced by Aspergillus sojae transformant As-penox2 strain To determine the physicochemical properties of PenOX2, the following enzyme activity measurement method was used. 600μL of any buffer, 3.99U / mL peroxidase dissolved in deionized water, 1.8mM 4-aminoantipyrine, 400μL of 2mM TOOS solution, and 150μL of deionized water were incubated at any temperature for 10 minutes, and then 50μL of enzyme solution stored on ice and 400μL of 2mM pentosidine solution dissolved in 100mM potassium phosphate buffer (pH8.0) incubated at any temperature for 10 minutes were added, stirred, and reacted at any temperature for 3 minutes. A during the reaction 555 The time course of the change in was measured using a spectrophotometer (U-3900, Hitachi High-Tech Science Corporation). 555 The amount of change was regarded as the activity value. Furthermore, the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute at 37° C. was defined as 1 unit (U) and calculated according to the following formula.

[0153] Activity (U / mL)={(ΔAs-ΔA0)×1.6×df}÷(39.2×0.5×0.05) ΔAs: A of the reaction solution per minute 555 Amount of change ΔA0: A per minute in the control experiment 555 Amount of change 1.6: Total volume of reaction solution (mL) df: dilution factor 39.2: Millimolar extinction coefficient of the quinone imine dye produced by the reaction (mM -1 ·cm -1 ) 0.5: Molar number of quinoneimine dyes produced by 1 mol of hydrogen peroxide 0.05: Volume of enzyme solution (mL)

[0154] The physicochemical properties of penox2 were as follows: (a) Optimal pH range Each buffer was prepared so that the final concentration was 50 mM citric acid-100 mM potassium phosphate buffer (pH 4.0-7.5), the final concentration was 100 mM potassium phosphate buffer (pH 6.5-8.0), and the final concentration was 100 mM glycine buffer (pH 8.0-11.0), and the enzyme reaction was carried out at each pH at a temperature of 37°C using these buffers. The results are shown in Figure 7. PenOX2 showed the highest activity at pH 7.5. In addition, since the activity value at pH 6.5-8.0 was 70% or more of the activity value at about pH 7.5 potassium phosphate buffer, it was determined that the optimal pH of PenOX2 is pH 6.5-8.0, and the most preferable optimal pH is pH 7.5. (b) Optimum temperature range The activity of PenOX2 was measured at various temperatures using a potassium phosphate buffer (pH 7.5) with a final concentration of 50 mM. The results are shown in FIG. 8. The temperature range in which PenOX2 showed 80% or more activity was 37°C to 50°C, compared to the activity at around 50°C, which was the temperature at which the highest activity was observed. From the above, it was determined that the optimal temperature range for PenOX2 was 37°C to 50°C. (c) Thermal stability The enzyme solution was treated at each temperature for 10 minutes, and the remaining activity was evaluated by measuring the activity at 37° C. using a potassium phosphate buffer with a final concentration of 100 mM (final pH 7.5 at the time of activity measurement). The results of thermal stability are shown in FIG. 9, and PenOX2 was stable up to about 30° C. (d) Stable pH range The remaining activity of PenOX2 was measured after treatment for 20 hours at 25°C using 100mM citric acid-200mM potassium phosphate buffer (pH 3.0-6.5), 200mM potassium phosphate buffer (pH 6.5-8.0), and 200mM glycine buffer (pH 8.0-10.0) at each pH. The results are shown in Figure 10. The pH range in which PenOX2 showed 90% or more activity compared to the activity of PenOX2 stored at 4°C was pH 4.5-7.5, and the pH range in which it showed 60% or more activity was pH 4.0-9.0. (e) Activity against pentosidine In the activity measurement method, the activity was measured at 37° C. using a potassium phosphate buffer with a final concentration of 50 mM (final pH 7.5 at the time of activity measurement), and the activity value (U / mL) was calculated using the above formula. The activity value was found to be 7.8 U / mL, and the specific activity was 29.1 U / mg (Bradford method). (f) Km value for pentosidine In the activity measurement method described above, activity was measured at various concentrations of the substrate pentosidine in a potassium phosphate buffer (pH 7.5) with a final concentration of 50 mM at 37°C, and the Michaelis constant (Km) was calculated from the Lineweaver-Burk plot. The results are shown in Figure 11. The Km value for pentosidine (free form) was found to be 0.070 mM. (g)Molecular weight The molecular weight was determined by SDS-PAGE according to the Laemmli method. Mini-PROTEAN TGX Stain-Free Precast Gels 4-20% (Bio-Rad) were used as electrophoresis gels, and Precision Plus Protein All Blue Prestained Protein Standards were used as molecular weight markers. The results are shown in Figure 12. The molecular weight of PenOX2 was approximately 80,000.

[0155] (Example 10) Measurement of pentosidine oxidase activity of enzymes with sequence homology to PenOX1 and PenOX2

[0156] As mentioned above, both PenOX1 and PenOX2 had pentosidine oxidase activity. When the amino acid sequence identity was examined using the BLAST program, the amino acid sequence homology between the two was 38.2%. Next, the following three enzymes were purchased, and pentosidine oxidase activity was examined using the above activity measurement method at a final concentration of 100 mM potassium phosphate buffer (pH 7.5) at 37°C. The amino acid sequence homology with each of PenOX1 and PenOX2 and the pentosidine oxidase activity were as follows. (a) Crotalus adamanteus Derived amino acid oxidase Type VI (Merck) (SEQ ID NO: 12) Molecular weight: 130,000 The amino acid sequence homology of this enzyme with PenOX1 and PenOX2 was 26.8% and 23.5%, respectively. The enzyme was diluted with deionized water to a concentration of 1 mg / mL (burette method) and used for activity measurement. The pentosidine oxidase activity of this enzyme was 0.555 (U / mL), and the specific activity was 0.555 (U / mg). (b) Crotalus atrox Amino acid oxidase Type I (Merck) (SEQ ID NO: 13) Molecular weight: 59,000 (calculated based on amino acid sequence) The amino acid sequence homology of this enzyme with PenOX1 and PenOX2 was 26.3% and 23.4%, respectively. 1 mg of enzyme powder was dissolved in 1 mL of deionized water and used for activity measurement. The pentosidine oxidase activity of this enzyme was 0.022 (U / mL), and the specific activity was 0.022 (U / mg) as a reference value. (c) Trichoderma Viride Lysine oxidase (Merck) (SEQ ID NO: 14) Molecular weight: 116,000 The amino acid sequence homology of this enzyme with PenOX1 and PenOX2 was 24.0% and 23.3%, respectively. 1 mg of enzyme powder was dissolved in 1 mL of deionized water and used for activity measurement. The pentosidine oxidase activity of this enzyme was 0.063 (U / mL), and the specific activity was 0.063 (U / mg) as a reference value. The sequence homology between the enzymes used in this example is shown in the table below. [Table 2]

[0157] Example 11: Heterologous recombinant expression of Escapin in Aspergillus sojae At the 5' end of the gene encoding mature Escapin Aspergillus A gene encoding a signal peptide of the genus was added to the vector, and the vector was expressed in heterologous recombinant form using the koji mold Aspergillus sojae as a host.

[0158] -Preparation of expression vector The mature Escapin gene sequence was that of the giant sea hare ( Aplysia California ) was used, with 1,554 base pairs (SEQ ID NO: 18) modified with codons for expression in Aspergillus oryzae based on the amino acid sequence of SEQ ID NO: 15 (the base sequence is SEQ ID NO: 17). The gene encoding the signal peptide used was the 69-base pair exon region of the gene encoding the signal peptide of cellulose dehydrogenase derived from Aspergillus oryzae (SEQ ID NO: 16). At the 5' end of the gene encoding mature Escapin Aspergillus In order to incorporate a gene encoding a signal peptide of the genus SEQ ID NO:16 linked to the 5' end of SEQ ID NO:18 (hereinafter referred to as gene sequence A) into a plasmid, a gene sequence consisting of 12 base pairs (SEQ ID NO:20) was added to the 5' end of gene sequence A and a gene sequence consisting of 12 base pairs (SEQ ID NO:21) was added to the 3' end of gene sequence A by artificial gene synthesis (hereinafter referred to as gene sequence A').

[0159] The expression cassette for expressing gene sequence A used was the promoter sequence Ptef (748 bp upstream of the tef1 gene, sequence number 7), which is the promoter sequence of the translation elongation factor gene tef1, and the terminator sequence Talp (800 bp downstream of the alp gene, sequence number 8), which is the terminator sequence of the alkaline protease gene alp, as a terminator.

[0160] The selection marker used was the pyrG3 gene (1,487 bp including 56 bp upstream, 896 bp coding region, and 535 bp downstream, SEQ ID NO: 9), which complements the uracil / uridine requirement and enables the introduction of multiple copies of the gene (see JP 2018-068292 A). These Ptef, Talp, and pyrG3 genes were derived from the Aspergillus sojae ( Aspergillus sojae The gene was obtained by PCR using the genomic DNA of the NBRC4239 strain as a template.

[0161] Next, an In-Fusion HD Cloning Kit (Clontech) was used to link the respective DNAs. For example, when linking Ptef to gene sequence A and Talp, a reverse primer of SEQ ID NO: 22 was used for Ptef, and a forward primer of SEQ ID NO: 23 was used for Talp, and a DNA fragment was amplified by PCR. At this time, the 5' end of gene sequence A' has a 15 bp sequence (CAT sequence complementary to the start codon ATG of AoCDHss and SEQ ID NO: 20) complementary to the 5' end of the reverse primer (SEQ ID NO: 22) for amplifying Ptef, and the 3' end of gene sequence A' has a 15 bp sequence (TGA sequence of the stop codon of Escapin and SEQ ID NO: 21) complementary to the 5' end of the forward primer (SEQ ID NO: 23) for amplifying Talp, so that linking between Ptef, gene sequence A, and Talp is possible by infusion reaction. In this manner, an expression vector p19-pG3-AoCDHss-Escapin was prepared in which Ptef-AoCDHss-Escapin-Talp-pyrG3, in which Ptef, AoCDHss, mature Escapin, Talp and pyrG3 were linked in that order, was inserted into the multicloning site of the pUC19 plasmid.

[0162] - Creation and cultivation of Aspergillus oryzae expression strains Using the transformation plasmid p19-pG3-AoCDHss-Escapin obtained above, a pyrG gene disruptant of Aspergillus sojae (a strain lacking 48 bp upstream, 896 bp coding region, and 240 bp downstream of the pyrG gene) was transformed by the protoplast PEG method, and two Aspergillus sojae transformed strains (AoCDHss-Escapin strains) in which multiple copies of the AoCDHss-Escapin expression cassette were inserted were obtained.

[0163] The obtained Aspergillus sojae transformant strain AoCDHss-Escapin was inoculated into 15 mL of PPY liquid medium (2% (w / v) pinedic, 1% (w / v) polypeptone, 0.5% (w / v) yeast extract, 0.5% (w / v) potassium dihydrogen phosphate, 0.05% (w / v) magnesium sulfate heptahydrate) in a 50 mL Erlenmeyer flask and cultured with shaking at 30°C for 4 to 5 days.

[0164] (Example 12) Measurement of pentosidine in combination with amino acid decomposition enzyme Preparation of a solution containing amino acid decomposition enzyme 1 (Escapin, SEQ ID NO: 15) The culture supernatant of the transformant obtained in Example 11 was concentrated using an ultrafiltration membrane (Amicon Ultra 15-30k, Merck). To the ice-cooled concentrated solution, ice-cooled saturated aqueous ammonium sulfate solution was added so that the ammonium sulfate concentration was 70%. The mixture was allowed to stand at 4°C for 2 hours, and then centrifuged (15,000 rpm, 4°C, 15 minutes) to collect the precipitate, which was then redissolved in 0.1 M potassium phosphate buffer (pH 6.8).

[0165] Amino acid decomposition enzyme solution 2 (Eastern diamondback rattlesnake ( Crotalus adamanteus Preparation of a solution containing L-amino acid oxidase (SEQ ID NO: 19) derived from L-Amino Acid Oxidase from the Eastern Diamondback Rattlesnake Crotalus adamanteus ) Type I (Merck) was dissolved in 0.1 M potassium phosphate buffer, pH 6.8, to a concentration of 1 mg / ml, and concentrated using an ultrafiltration membrane (Amicon Ultra 15-30k, Merck).

[0166] Preparation of enzyme solution for pentosidine measurement The A. sojae recombinant strain As-penox2 obtained in the sections "Preparation of expression vector" and "Preparation and cultivation of koji mold expression strain" in Example 5 was cultured in sterilized PPY medium at 180 rpm and 30°C for 5 days with shaking. The resulting culture supernatant was concentrated using an ultrafiltration membrane (Amicon Ultra 15-30k, Merck). Ammonium sulfate was gradually added to the concentrated solution to 70% saturation, and the solution was left at 4°C for 2 hours, then centrifuged (15,000 rpm, 4°C, 15 minutes) to recover the supernatant. The recovered supernatant was concentrated using an ultrafiltration membrane (Amicon Ultra 0.5-30k, Merck) and replaced with 0.1 M potassium phosphate buffer pH 6.8.

[0167] - Contaminant elimination test for pentosidine measurement In a system for measuring pentosidine using pentosidine oxidase, a model system in which various amino acids were artificially added as impurities to the solution to be measured was used to verify the effect of the measurement method of the present invention in measuring pentosidine.

[0168] (1) Preparation of pentosidine solution Pentosidine (free form equivalent) (Peptide Institute, 3TFA salt was used) was dissolved in deionized water to a concentration of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, or 0.5 mM. (2) Preparation of impurity solution The components were dissolved in 0.1 M potassium phosphate buffer, pH 6.8, to give final concentrations of 290 μM L-alanine, 48 μM L-cysteine, 4 μM L-aspartic acid, 57 μM L-glutamic acid, 40 μM L-phenylalanine, 245 μM glycine, 54 μM L-isoleucine, 128 μM L-lysine, 92 μM L-leucine, 22 μM L-methionine, 184 μM L-proline, 60 μM L-arginine, 94 μM L-serine, 116 μM L-threonine, 155 μM L-valine, 39 μM L-tryptophan, and 45 μM L-tyrosine. The amino acid concentrations were set based on the literature values ​​(Mayo Clinic Laboratories Neurology Catalog's "Plasma Amino Acid Reference Values" (https: / / neurology.testcatalog.org / show / AAQP, accessed October 10, 2018; https: / / www.mayomedicallaboratories.com / test-catalog / Clinical+and+Interpretive / 9265, accessed June 24, 2020) for each amino acid amount in the blood of people aged 18 years or older (half the upper limit). (3) Preparation of reagents The reagents used in the measurement were prepared as follows. 3A. Color Reagent Peroxidase (manufactured by Toyobo) was dissolved in deionized water to a concentration of 3.99 U / ml, 4-aminoantipyrine (manufactured by Fluka) to 1.8 mM, and TOOS (manufactured by Dojindo) to 2 mM. 3B. Contaminant removal reagent Amino acid decomposition enzyme solution 1 (1.63 U / ml) and amino acid decomposition enzyme solution 2 (2.40 U / ml) were mixed in a liquid volume ratio of 5:3. 3C. Pentosidine measurement reagents An enzyme solution (3.31 U / ml) for measuring pentosidine was used.

[0169] (4) Measurement All measurements were performed at room temperature unless otherwise specified. A 96-well microwell plate (manufactured by Nunc) was used for the reaction. 20 μl of the impurity solution (2) above, 25 μl of the color-developing reagent (3A) above, and 50 μl of the impurity elimination reagent (3B) above were added to 5 μl of the pentosidine solution (1) above in that order, and the absorbance at 555 nm after 10 minutes was measured, which was taken as data 1. Next, 25 μl of the pentosidine measurement reagent (3C) above was added, and the absorbance at 555 nm was measured after 10 minutes, which was taken as Data 2. The value (ΔOD) obtained by subtracting Data 1 from Data 2 was taken as the measured value. As Comparative Example 1, a solution containing no impurities, i.e., a solution in which the impurity solution (2) above was replaced with 0.1 M potassium phosphate buffer, pH 6.8, was also measured. As Comparative Example 2, a system containing impurities but not removing the impurities, that is, a solution in which the impurity removal reagent in (3B) above was replaced with 0.1 M potassium phosphate buffer, pH 6.8, was also measured. The average value of three measurements is shown in the following Table 3. Furthermore, the relative values ​​of Comparative Example 2 and the Example, with the measured value of Comparative Example 1 being taken as 100%, are shown in Table 4.

[0170] [Table 3] [Table 4]

[0171] As shown in the above table, in the pentosidine measurement without removing impurities (Comparative Example 2), the pentosidine concentration was measured to be significantly higher than that in the measurement without adding impurities (Comparative Example 1), reflecting the addition of impurities, and an accurate value was not obtained. In contrast, in measurements including an elimination process using an impurity elimination reagent, the measurement value due to impurities was small, and a value close to the pentosidine concentration obtained in the system in Comparative Example 1 in which no impurities were added was obtained, thereby reducing the influence of impurities that cause measurement errors and enabling accurate measurements.

[0172] (Example 13) Substrate specificity analysis of amino acid decomposition enzyme 1 The substrate specificity of amino acid elimination enzyme 1 for various amino acids and pentosidine was analyzed.

[0173] (1) Preparation of enzyme solution The amino acid elimination enzyme solution 1 obtained in Example 12 was diluted with 0.1 M potassium phosphate buffer, pH 6.8, to a concentration of 0.134 U / ml. (2) Preparation of various amino acid and pentosidine solutions Pentosidine (as in Example 12) was dissolved in deionized water to a concentration of 2 mM. Various amino acids were dissolved in deionized water to a concentration of 4 mM. (3) Preparation of color-developing reagent The solutions were dissolved in deionized water to a concentration of 3.99 U / ml peroxidase (manufactured by TOYOBO), 1.8 mM 4-aminoantipyrine (manufactured by Fluka), and 2 mM TOOS (manufactured by Dojindo).

[0174] (4) Measurement All measurements were carried out at room temperature unless otherwise specified. A 96-well microwell plate (manufactured by Nunc) was used for the reaction. To 50 μl of the enzyme solution (1) above, 25 μl of the various amino acid or pentosidine solution (2) above and 25 μl of the color-developing reagent (3) above were added in that order, and the absorbance at 555 nm was measured at the start and after 10 minutes. The slope of the increase in absorbance was regarded as the reaction rate for the target substrate. The reaction rates for various amino acids and pentosidine, i.e., substrate specificity, relative to the reaction rate for arginine, the substrate with the highest reaction rate, set at 100, are shown in FIG.

[0175] (Example 14) Substrate specificity analysis of amino acid elimination enzyme 2 The substrate specificity of amino acid elimination enzyme 2 for various amino acids and pentosidine was analyzed.

[0176] (1) Preparation of enzyme solution The amino acid elimination enzyme solution 2 obtained in Example 12 was diluted with 0.1 M potassium phosphate buffer, pH 6.8, to a concentration of 0.12 U / ml. (2) Various amino acid and pentosidine solutions and (3) color-developing reagent were prepared in the same manner as in Example 13.

[0177] (4) Measurement All measurements were performed at room temperature unless otherwise specified. A 96-well microwell plate (manufactured by Nunc) was used for the reaction. 25 μl of the various amino acids or pentosidine solution (2) above and 25 μl of the color-developing reagent (3) above were added to 50 μl of the enzyme solution (1) above in that order, and the absorbance at 555 nm was measured at the start and after 10 minutes, and the slope of the increase in absorbance was considered to be the reaction rate for the target substrate. The reaction rates for various amino acids and pentosidine, i.e., substrate specificity, when the reaction rate for leucine, which had the highest reaction rate, was set to 100, are shown in FIG. 14.

[0178] (Example 15) Substrate specificity analysis of enzyme for measuring pentosidine The substrate specificity of the enzyme for measuring pentosidine for various amino acids and pentosidine was analyzed.

[0179] (1) Preparation of enzyme solution The enzyme solution for measuring pentosidine obtained in Example 12 was diluted with 0.1 M potassium phosphate buffer, pH 6.8, to a concentration of 0.083 U / ml. (2) Various amino acid and pentosidine solutions and (3) color-developing reagent were prepared in the same manner as in Example 13.

[0180] (4) Measurement All measurements were performed at room temperature unless otherwise specified. A 96-well microwell plate (manufactured by Nunc) was used for the reaction. 25 μl of the various amino acids or pentosidine solution (2) above and 25 μl of the color-developing reagent (3) above were added to 50 μl of the enzyme solution (1) above in that order, and the absorbance at 555 nm was measured at the start and after 10 minutes, and the slope of the increase in absorbance was regarded as the reaction rate for the target substrate. The reaction rates for various amino acids, i.e., substrate specificity, when the reaction rate for pentosidine is set at 100, are shown in FIG.

[0181] From the results of Examples 13 to 15, it was considered that in Example 12, amino acids highly reactive with the enzyme for measuring pentosidine were eliminated by the amino acid decomposition enzyme 1 solution and the amino acid decomposition enzyme 2 solution, and the influence of impurities that cause measurement errors was reduced, enabling accurate measurement of pentosidine.

Claims

1. A method for measuring pentosidine in a sample, comprising: Decomposing the sample with an amino acid decomposing enzyme; contacting the sample after the decomposition step with pentosidine oxidase; and detecting a change caused by said contact; The amino acid decomposition enzyme and the pentosidine oxidase are different, The pentosidine oxidase is selected from the group consisting of the following (a) to (d): (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (b) a protein encoded by a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; (c) a protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (d) a protein encoded by a gene consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; The measurement method of claim 1, wherein the protein is any protein selected from the group consisting of:

2. The method according to claim 1 , wherein a change in the amount of oxygen, hydrogen peroxide or ammonia is detected in the detection step.

3. The pentosidine oxidase has the following physicochemical properties: (1) Action: Activity of oxidatively decomposing pentosidine; and (2) Molecular weight by SDS-PAGE: 75,000 to 85,000 The method according to claim 1 or 2, comprising:

4. The method according to any one of claims 1 to 3, wherein the pentosidine oxidase is derived from a filamentous fungus.

5. The method according to any one of claims 1 to 4, wherein the amino acid decomposing enzyme decomposes amino acids contained in a sample, and the amino acids are selected from the group consisting of arginine, leucine, methionine, phenylalanine, tryptophan, and tyrosine.

6. The method according to any one of claims 1 to 5, wherein the amino acid decomposition enzyme is selected from the group consisting of amino acid oxidase, amino acid dehydrogenase, amino acid aminotransferase, amino acid decarboxylase, amino acid ammonia lyase, amino acid oxygenase and amino acid hydrolase.

7. (i) an amino acid degrading enzyme; and (ii) pentosidine oxidase Including, The pentosidine oxidase is selected from the group consisting of the following (a) to (d): (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (b) a protein encoded by a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; (c) a protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (d) a protein encoded by a gene consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; Any protein selected from the group consisting of: A kit for measuring pentosidine in samples.

8. The kit according to claim 7 , wherein the amino acid decomposition enzyme is an enzyme that decomposes an amino acid selected from the group consisting of arginine, leucine, methionine, phenylalanine, tryptophan and tyrosine.

9. A method for producing a reaction product of pentosidine derived from a sample, comprising: Decomposing the sample with an amino acid decomposing enzyme; contacting the specimen after the decomposition step with pentosidine oxidase The pentosidine oxidase is selected from the group consisting of the following (a) to (d): (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (b) a protein encoded by a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; (c) a protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (d) a protein encoded by a gene consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6; Any protein selected from the group consisting of: wherein the amino acid degrading enzyme and the pentosidine oxidase are different.

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