JP7216968B2 - Amino acid quantification method and amino acid quantification kit - Google Patents

Description

The present invention relates to an amino acid quantification method, an amino acid quantification kit, and the like.

Amino acids play important roles as constituents of proteins in living organisms. Many studies have been made on the functionality of amino acids, and amino acids are used in various industries such as pharmaceuticals, processed foods, and health foods. For example, free amino acids in food are related to taste, flavor after heating, preservability, bioregulatory function after ingestion, etc., and are attracting attention as important factors in the fields of food science and nutritional science. In recent years, it has been found that the amino acid concentration in the blood changes due to disease, and by measuring the amino acid concentration in the blood, it is also used as a biomarker that enables cancer diagnosis such as lung cancer, stomach cancer, and colon cancer. It is utilized. Therefore, amino acid quantification technology has become an indispensable technology in various fields such as product development, quality control, and diagnosis using amino acids.

As an amino acid quantification technique, a method is known in which amino acids are separated by liquid chromatography and detected by color reaction with ninhydrin or orthophthalaldehyde (Non-Patent Document 1). However, this method has the problem that it takes about 2 hours to analyze one specimen, and thus it is not suitable for measuring a large number of specimens.

As another amino acid quantification technique, a method for measuring amino acids using an enzyme that acts on amino acids is known (Patent Document 1). However, this method has the problem that it has low selectivity for the desired amino acid as a substrate and also reacts with amino acids other than the desired substrate.

Aminoacyl-tRNA synthetase (AARS) is an enzyme involved in protein synthesis in vivo, and there are 20 types of AARS specific to 20 types of amino acids. Therefore, it is considered that the enzyme has extremely high selectivity for target amino acids, and an amino acid quantification technique using a reaction involving AARS (AARS reaction) has been developed so far. For example, Patent Document 2 describes a method for analyzing amino acids using AARS. In this method, AARS functions as a catalyst to react ATP and amino acid molecules one by one to produce aminoacyl AMP and pyrophosphate, and aminoacids are analyzed using the produced pyrophosphate as an indicator. However, in this method, AARS reacts with one molecule of amino acid and ATP to produce one molecule of aminoacyl AMP, so only the same amount of pyrophosphate as the amino acid in the sample or less is produced (Non-Patent Documents 2 and 3). , 4).

Furthermore, a method for quantifying amino acids based on the AARS reaction is described in Patent Document 3 mentioned above. That is, as described above, aminoacyl AMP normally binds strongly to AARS to form an aminoacyl AMP-AARS complex. Therefore, in this method, by adding an amine (nucleophilic agent) such as hydroxylamine as an aminoacyl AMP-AARS complex decomposing reagent, the AARS is made to be in a state capable of reacting again, and as a result, an amino acid can be decomposed with a small amount of AARS. It is characterized by quantifying. However, in the method described in Patent Document 3, the complex-dissolving reagent reacts with the amino acid of the complex to produce a compound (for example, as shown in paragraph 0037 of Patent Document 3, the complex-dissolving When hydroxylamine is used as a reagent, "amino acid hydroxamic acid" is produced), amino acids cannot be reused, and as a result, only pyrophosphate, which is a product equivalent to the amino acids in the sample, is produced. It cannot be obtained (Paragraph No. 0023 of Patent Document 3).

As described above, the method involving AARS has a problem in high-sensitivity analysis because the amount of pyrophosphate produced is small. Furthermore, there are 20 types of AARS that are specific to 20 types of amino acids, but there is also a problem that there is no AARS that reacts with other than 20 types of amino acids such as ornithine.

Non-ribosomal peptide synthetase (NRPS) is an enzyme involved in complex peptide synthesis and is used for the synthesis of antibiotics and the like. NRPS acts on proteinogenic amino acids (20 types of L-type amino acids that constitute proteins), and also isomers of D-type amino acids, as well as modified amino acids such as methylation, acylation, and glycosylation, ornithine, and the like. It also acts on non-protein amino acids (amino acids that are not structural units of proteins), and is said to be an enzyme with extremely high selectivity (specificity). NRPS is composed of three domains: an adenylation domain (A domain), a thiolation domain (T domain), and a condensation domain (C domain). The NRPS reaction is as shown in FIG. 1. In the A domain, aminoacyl adenylic acid (aminoacyl AMP) and pyrophosphate (PPi ) is produced. In the T domain, aminoacyl AMP then reacts with the aminoacyl 4'phosphopantetheine group (4'PP) of the T domain to form an aminoacyl-T domain complex. Subsequently, in the C domain, a peptide is produced by causing a peptide condensation reaction that bonds the amino acids bound to the aminoacyl-T domain complex. NRPS synthesizes a linear or cyclic peptide consisting of 3- to 15-mer amino acids by repeating such reactions (Non-Patent Document 5, Non-Patent Document 6).

Techniques described in known documents (Patent Documents 4 and 5, Non-Patent Documents 5 and 6) relating to NRPS relate to peptide synthesis by NRPS consisting of A, T and C domains, wherein the A domain or the A domain and a T domain, and does not aim to solve technical problems related to amino acid quantification by NRPS. In addition, in the known literature on NRPS (Non-Patent Document 7), there is a description of a method for measuring the activity of NRPS used for biopolymer synthesis. Since the AMP-A domain complex is formed, it reacts only up to the concentration of NRPS in the sample. It is now in a state where it can react again, and the activity of NRPS is being confirmed. However, in this method, the complex-dissolving reagent reacts with the amino acid of the complex to produce a compound, and the amino acid cannot be reused. only pyrophosphate is produced. These known documents make no mention of amino acid quantification utilizing the NRPS reaction and the use of an NRPS consisting of an A domain or an A domain and a T domain for amino acid quantification. Nor is there any mention of the recycling of amino acids and NRPS in reactions with NRPS.

JP 2013-146264 A JP 2011-50357 A Patent No. 5305208 WO2006/001382 Patent No. 5367272

"Chemistry and Biology", (Japan), 2015, Vol.53, p. 192-197 "Akimitsu Kugimiya, Department of Creative Science, Graduate School of Information Science, Hiroshima City University" [online], Hiroshima City University website, [Searched 2016/01/13], Internet (URL: http://rsw.office.hiroshima-cu.ac. jp/Profiles/2/000129/profile.html) Analytical Biochem., 443, 22-26, 2013 Appl. Biochem. Biotechnol., 174, 2527-2536, 2014 "The Chemical Society of Japan NEWS LETTER", (Japan), 2015, Vol.29, p. 3-6 "Chemistry and Biology", (Japan), 2006, Vol.44, p. 85-92 Analytical Sciences January, 30, 17-24, 2014

The present invention solves various problems in the prior art related to the above-described amino acid quantification method, and provides proteinogenic amino acids to be measured (20 L-type amino acids that constitute proteins), D-type amino acids, modified An object of the present invention is to provide a method and a kit for amino acid quantification that are specific, simple, and highly sensitive to quantify amino acids and non-protein amino acids such as ornithine.

In the method of quantifying the amount of amino acids in a sample using NRPS, in the conventional NRPS reaction consisting of A, T and C domains, amino acids and ATP react with NRPS to produce peptides. It is thought that the amino acids cannot be reused and that the amount of pyrophosphate that is equal to or less than the amino acids is produced. In addition, in the NRPS reaction consisting of A and T domains, one molecule of NRPS reacts with one amino acid molecule and ATP, but since an aminoacyl-T domain complex is formed, the amino acids in the sample cannot be reused, It is believed that as much pyrophosphate as or less than the NRPS in the sample is produced. Furthermore, in the NRPS reaction consisting of the A domain, one molecule of NRPS reacts with one molecule of amino acid and ATP. It is believed that only acid is produced. As a result of various studies, the inventors released NRPS and amino acid (AA) from the once-formed aminoacyl AMP-NRPS complex, as shown in FIG. It was found that by using the compound to form a complex, a reaction product such as pyrophosphate, which is the object of measurement, can be produced to a higher molar number than the NRPS and/or amino acids contained in the sample, I completed the present invention.

The present invention relates to the following aspects [1] to [6].
[1] Step (I) including the following steps:
(Step I-1) In the presence of divalent cations, an amino acid (AA) in a sample, a non-ribosomal peptide synthetase (NRPS) corresponding to the AA, and adenosine triphosphate (ATP) are reacted. , a reaction (reaction 1) to form a conjugate consisting of aminoacyl adenylic acid (aminoacyl AMP) and NRPS (aminoacyl AMP-NRPS conjugate);
(Step I-2) A step including a reaction (Reaction 2) in which an aminoacyl AMP-NRPS complex formed in Reaction 1 and/or Reaction 3 is reacted with an amino acid regenerating reagent to liberate NRPS and AA from the complex. ;
(Step I-3) a step comprising a reaction (reaction 3) to form an aminoacyl AMP-NRPS conjugate reaction by recycling the AA and/or NRPS liberated in reaction 2 in reaction 1;
(Step I-4) repeating steps I-2 and I-3, and
step (II) comprising measuring the amount of the reaction product generated in step (I) and determining the amount of the amino acid based on the measured amount of the reaction product;
and wherein said NRPS comprises at least an A domain.
[2] The method for quantifying amino acids according to [1], wherein the NRPS consists of an A domain and/or an A domain and a T domain.
[3] The method for quantifying amino acids according to [1], wherein the amino acid regeneration reagent used in step (I) is a nucleotide and/or an alkaline compound.
[4] The method for quantifying amino acids according to any one of [1] to [3], wherein the amount of the reaction product produced in step (I) is measured by measuring absorbance change by an absorbance method.
[5] The method for quantifying amino acids according to any one of [1] to [4], wherein at least one of pyrophosphate and hydrogen ions is measured as the reaction product generated in step (I).
[6] Any one of [1] to [5], wherein the number of moles of the reaction product produced in step (I) is greater than the number of moles of NRPS and/or amino acids in the sample. Amino acid quantification method.
[7] An amino acid quantification kit for performing the amino acid quantification method according to [1] to [6], comprising ATP, a nucleotide, and an NRPS corresponding to the amino acid.

In the amino acid quantification method according to the present invention, proteinogenic amino acids (20 types of L-type amino acids that constitute proteins) can be specifically measured, and isomers of D-type amino acids, methylation, Modified amino acids such as acylation, glycosylation and non-proteinogenic amino acids such as ornithine can also be specifically measured. In addition, by liberating NRPS and amino acids from the formed aminoacyl AMP-NRPS conjugate and repeatedly using them to form the aminoacyl AMP-NRPS conjugate, reaction products such as pyrophosphate to be measured can be obtained from the sample. Up to more moles than the NRPS and/or amino acids contained therein can be produced. As a result, even when these reaction products are measured by means that are simpler than those of the conventional techniques, the conventional techniques for amino acid quantification using high-sensitivity analytical methods such as fluorescence methods using multi-step enzymatic reactions have been proposed. It becomes possible to quantify in a range equivalent to the range. Furthermore, when using the same measurement means, it is possible to quantify amino acids in a lower concentration range.

Furthermore, according to the present invention, a method for specifically, simply, and highly sensitively quantifying proteinogenic amino acids to be measured, as well as non-proteinogenic amino acids such as D-amino acids, modified amino acids and ornithine, using NRPS, and for amino acid quantification We can provide a kit.

FIG. 3 shows an NRPS reaction; It is a figure which shows the reaction process of this invention. FIG. 4 is a diagram showing the amount of pyrophosphate produced by various NRPS reactions. FIG. 4 is a diagram showing the amount of pyrophosphate produced by various ATP and enzyme concentrations. FIG. 4 is a diagram showing the amount of pyrophosphate produced depending on the type of divalent cation. FIG. 4 is a diagram showing the amount of pyrophosphate produced at various divalent cation concentrations. It is a figure which shows the pyrophosphate production amount in each pH. It is a figure which shows the pyrophosphate production amount in each temperature. FIG. 10 is a diagram showing the amount of pyrophosphate produced in the NRPS reaction of the present invention and a known document (Non-Patent Document 7). FIG. 4 shows an amino acid calibration curve in pyrophosphate measurement by the molybdenum blue method.

In Reaction 1 and Reaction 3 of the method of the present invention, an amino acid, NRPS corresponding to the amino acid, and ATP are reacted to form an aminoacyl AMP-NRPS conjugate. Any NRPS known to those skilled in the art that acts specifically on each amino acid can be used as the NRPS used in the method of the present invention. For example, ornithine (Orn) includes NRPS that specifically acts on ornithine, and lysine (Lys) includes NRPS that specifically acts on lysine. The NRPS used in the present invention may be any NRPS that is derived from various organisms, such as those derived from microorganisms such as Streptomyces, Corynebacterium, Pseudomonas, Aspergillus, Saccharopolyspora, and Lecanicillum. NRPS derived from microorganisms is preferred, particularly from the viewpoint of handling and productivity. The NRPS is composed of an A domain, a T domain and a C domain, preferably composed of only the A domain and/or composed of the A domain and the T domain. Alternatively, it may be a recombinant NRPS or a synthetic NRPS. Soluble enzymes are preferred, but surfactants may be combined with insoluble enzymes, and enzymes obtained by solubilizing insoluble enzymes by fusion with solubilizing proteins or deletion of membrane-binding portions may be used. The known amino acid sequence of NRPS can be used, and the recombinant NRPS composed of the A domain has a sequence with 60%, 70%, 80%, 90% or 95% or more identity, and A recombinant NRPS composed of an A domain and a T domain has a sequence with 60%, 70%, 80%, 90% or 95% or more identity and has NRPS activity. Also good.

The NRPS used in the present invention can be prepared by any method or means known to those skilled in the art, for example, water is added to an object containing NRPS, crushed with a crusher, an ultrasonic crusher, etc., and then crushed. An extract obtained by removing solid matter by centrifugation, filtration, or the like, and NRPS obtained by further purifying and isolating the extract by column chromatography or the like can be used. That is, the main technical feature of the present invention is that, in a method for quantifying amino acids using NRPS, NRPS and amino acids are liberated from the formed aminoacyl AMP-NRPS complex, and they are converted into an aminoacyl AMP-NRPS complex. By repeatedly using it for formation, the reaction product such as pyrophosphate to be measured is produced to a larger number of moles than the NRPS and/or amino acids contained in the sample, and the method for preparing NRPS is not limited at all. not to be

Amino acids to be measured in the present invention include 20 types of L-type proteinogenic amino acids such as L-lysine, L-leucine and L-isoleucine, and L-type amino acid isomers D-lysine, D-leucine and D - D-form amino acids such as isoleucine, non-proteinogenic amino acids such as ornithine, β-lysine and α-methyl-L serine.

Moreover, the sample used in the present invention is not particularly limited, and examples thereof include blood, fresh foods, processed foods and beverages. The amino acid concentration in each sample is different for each amino acid. For example, the amino acid concentration in blood is 41-85 nmol/mL for isoleucine, 81-154 nmol/mL for leucine, 158-288 nmol/mL for valine, 119-257 nmol/mL for lysine, and 43-257 nmol/mL for ornithine. such as 96 nmol/mL. The content of free amino acids in fresh foods, for example, in garlic, is 5 mg/100 g for lysine and 136 mg/100 g for arginine. The content of free amino acids in processed foods and beverages, for example, in soy sauce, is 213 mg/100 g for lysine, 348 mg/100 g for alanine, and 450 mg/100 g for leucine. For fully ripe Robusta coffee beans, aspartic acid is 76 mg/100 g, leucine is 6 mg/100 g, valine is 10 mg/100 g, and so on. Depending on the expected amino acid concentration in each of these samples, appropriate dilutions can be prepared and used as the samples of the present invention.

Amino acids in a sample used in the present invention may be in a mixture of L- and D-forms. In such a case, for example, it is preferable to remove either the L- or D-amino acid as a pretreatment in the method of the present invention. Methods for removing either the L or D amino acid include any method known to those skilled in the art, such as removal of either the L or D amino acid by column chromatography or an appropriate enzyme. . In addition, although L- and D-forms are mixed in biological samples, most D-form amino acids in mammals are about 0.1 to 1.0% of L-form amino acids, and in fruits, D-form amino acids are It is 3.0% or less of the L-amino acid, and the D-amino acid content in the biological sample does not affect the determination of the L-amino acid.

The NRPS concentration in the reaction solution used for the reaction can be appropriately determined by a person skilled in the art according to the type of sample, the estimated amino acid concentration in the sample, the ATP concentration, and various reaction conditions such as reaction time and temperature. be done. By increasing the NRPS concentration, the reaction can be completed in a short time. Conversely, if a long reaction time is acceptable, the NRPS concentration may be low. For example, the concentration of NRPS derived from actinomycetes such as Streptomyces, Saccharopolyspora and Corynebacterium is 0.1 μM or more, more preferably 0.5 μM or more, still more preferably 1.0 μM or more, particularly preferably 5.0 μM or more, Most preferably, it can be 10.0 μM or more. In any case, the method of the present invention has the advantage that NRPS is used repeatedly, so that it is not necessary to add an excess amount of NRPS relative to the expected amount of amino acids in the sample. Therefore, the upper limit of the NRPS concentration can be appropriately set by a person skilled in the art in consideration of economic efficiency and the like.

ATP and divalent cations are used together with NRPS in Reaction 1 or Reaction 3 of the method of the present invention. ATP used in the present invention may be sodium salt, lithium salt, or the like. The concentration of ATP in the reaction solution used in the reaction depends on the type of sample, expected amino acid concentration, NRPS concentration, nucleotide concentration in the sample, and various reaction conditions such as reaction time and temperature. The manufacturer can appropriately determine the amount, but it is preferable to add the amino acid in an excess amount relative to the expected amino acid concentration in the sample. For example, when the sample is blood, the ATP concentration should be 0.01 mM or higher, more preferably 0.1 mM or higher, still more preferably 1.0 mM or higher, particularly preferably 10.0 mM or higher, and most preferably 40.0 mM or higher. can be done. The upper limit of the ATP concentration can be appropriately set by those skilled in the art in consideration of economic efficiency and the like. Divalent cations used in the present invention can be magnesium, manganese, cobalt, calcium and the like. Since divalent cations have different requirements depending on the NRPS, divalent cations suitable for the NRPS to be used may be appropriately used. The concentration of the divalent cation in the reaction solution used for the reaction can be determined appropriately, but it is preferable to add the divalent cation in an amount equal to or higher than the ATP concentration. For example, the ATP: divalent cation ratio in NRPS is at least 1:10, more preferably at least 1:7, even more preferably at least 1:5, particularly preferably at least 1:3, most preferably at least 1:1. can be

Subsequently, in Reaction 2 of the method of the present invention, an amino acid regeneration reagent acts on the aminoacyl AMP-NRPS complex formed in Reaction 1 and/or Reaction 3 to liberate NRPS and amino acids from the complex. The amino acid regeneration reagent for the reaction is arbitrarily selected from ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), deoxyadenosine triphosphate (dATP), and the like. Nucleotides consisting of one type or a combination thereof, and alkaline compounds that generate hydroxide ions such as sodium hydroxide, potassium hydroxide, sodium carbonate and buffering agents can be used. In Reaction 2, NRPS and amino acids are liberated from the aminoacyl AMP-NRPS conjugate with the formation of AMP, Ap4A, etc., depending on the amino acid regenerating reagent used. The concentration of the amino acid regeneration reagent in the reaction solution used in the reaction depends on various reaction conditions such as the type of sample, expected amino acid concentration in the sample, NRPS concentration, ATP concentration, and reaction time/temperature. Although determined by the manufacturer, it is preferable to add nucleotides in an excess amount relative to the concentration of amino acids in the sample because they are consumed in the production of AMP, Ap4A, and the like. For example, when the sample is blood, the nucleotide concentration is 0.01 mM or higher, more preferably 0.1 mM or higher, still more preferably 1.0 mM or higher, particularly preferably 10.0 mM or higher, and most preferably 40.0 mM or higher. be able to. Further, the alkaline compound should be able to make the reaction field (reaction system) pH 7 or higher. For example, for NRPS derived from actinomycetes such as Streptomyces, Saccharopolyspora and Corynebacterium, the pH is preferably 7.0 or higher, more preferably 7.5 or higher, and still more preferably 8.0 or higher. The reaction pH can be adjusted using any buffer known to those skilled in the art, such as HEPES buffer, CHES buffer, TRIS buffer, MOPS buffer, and the like. Therefore, in the method of the present invention, there is no need to add an aminoacyl AMP-NRPS complex decomposing reagent such as amines as described in Patent Document 3 in order to restore the NRPS to a state capable of reacting. Since the released amino acid does not react with the reagent, the released amino acid can be reused to form the aminoacyl AMP-NRPS conjugate.

In Reaction 3 of the method of the present invention, the amino acid and/or NRPS liberated in Reaction 2 are reused in Reaction 1 to form an aminoacyl AMP-NRPS conjugate reaction. Furthermore, in step (I) of the method of the present invention, the pyrophosphate generated in Reaction 1 or 3 and AMP in the reaction system are reacted with phosphoenolpyruvate and pyruvate dikinase by any method known to those skilled in the art. and/or Ap4A pyrophosphorus to Ap4A generated from nucleotides in Reaction 2. ADP produced by the action of hydrolase can also be reused as a nucleotide in Reaction 2.

For example, if ATP is selected as the nucleotide that acts in reaction 2 and the reaction as described above is not used to regenerate ATP, step (I ), it is preferable to add a higher concentration of ATP, which is the sum of the respective concentrations of ATP and nucleotides shown above, to the entire reaction system.

As a result of the above, under the reaction conditions such as the above-mentioned amino acid regeneration reagent (nucleotide such as ATP and/or alkaline compound) and the composition of NRPS corresponding to the amino acid, the result of the reaction in step (I) was pyrophosphate and/or Or for each of the respective reaction products, such as hydrogen ions, more moles of molecules can be produced than the moles of NRPS and/or amino acid to be measured contained in the sample. As a result, the method of the present invention enables quantification of amino acids at concentrations much lower than those of the prior art. Therefore, this point can be said to be a remarkable effect of the present invention as compared with the prior art.

However, even if the reaction product such as pyrophosphate produced under the reaction conditions is in an amount equal to or less than the number of moles of amino acids in the sample, if the amino acid can be quantified based on the reaction product, pyroline It is not necessary that reaction products such as acids be produced in excess of the number of moles of the amino acid. Therefore, the amounts of pyrophosphate and hydrogen ions produced in step (I) of the present invention are as long as amino acids can be quantified by an appropriate method for measuring the reaction product in step (II). It is not particularly limited. In addition, regarding the number of repetitions of Reaction 2 (Step I-2) and Reaction 3 (Step I-3) in (Step I-4) of the method of the present invention, the reaction product in Step (II) There is no particular limitation as long as the amino acids can be quantified by an appropriate measuring method for living organisms.

The reaction temperature in step (I) of the process of the present invention may be any temperature at which each reaction occurs. For example, in the case of NRPS derived from actinomycetes such as Streptomyces, Saccharopolyspora and Corynebacterium, the temperature is preferably 4 to 50°C, more preferably 10 to 40°C, and most preferably 20 to 35°C. In addition, the reaction time may be any time that causes the NRPS reaction with the amino acid in the sample, preferably about 5 to 120 minutes, more preferably about 10 to 90 minutes, and still more preferably about 15 to 60 minutes. is desirable.

Reaction components such as reagents and enzymes used in each step included in step (I) of the method of the present invention can be added to the reaction system by any means, procedures, etc. known to those skilled in the art, as long as the addition method causes the NRPS reaction. can be added to For example, each component may be added to the reaction system at once before starting the reaction, or the NRPS or amino acid may be added last and allowed to react.

In step (II) of the method of the present invention, the amount of each reaction product such as pyrophosphate and hydrogen ion produced in step (I) is measured, and the amount of amino acid is determined based on the measured amount of the reaction product. do. In step (II), the reaction between the amino acids in the sample in step (I) and NRPS is performed, depending on the measurement method, for example, by adding trichloroacetic acid to the reaction system as described in the Examples. After terminating the reaction by any method or means known to those skilled in the art, or at any stage during the reaction in step (I), the reaction can be carried out as appropriate.

Any method or means known to those skilled in the art can be used to measure the amount of pyrophosphate produced in step (I) of the present invention, for example, measuring the absorbance of a blue complex produced by reacting molybdic acid with pyrophosphate. Pyrophosphate can be measured by the Blue method, a method of combining hypoxanthine-guanine phosphoribosyltransferase, xanthine oxidase or xanthine dehydrogenase, a method of combining luminol with inorganic pyrophosphatase, pyruvate oxidase and peroxidase and measuring the luminescence of the product. An enzymatic method or the like can be used. Alternatively, a measurement method may be used in which an oxidation-reduction reaction is caused by an enzyme reaction or the like, and a potential change is measured by a multi-electrode potential measuring instrument that detects a current value derived from the oxidation-reduction reaction. Furthermore, for the measurement of hydrogen ions produced in the reaction, a measurement method of measuring a potential change using a glass electrode for detecting hydrogen ions or an ion-sensitive field effect transistor, or the like can be used. Pyrophosphate, hydrogen ions, and the like generated in step (I) of the present invention can be separated from the reaction solution and measured. The method for separating pyrophosphate, hydrogen ions, etc. from the reaction solution is not particularly limited as long as it is a method that does not affect the measurement. For example, protein removal by acid treatment, paper chromatography separation, separation with a microfluidic device, etc. is mentioned. The main technical feature of the present invention is that, in a method for quantifying amino acids using NRPS, NRPS and amino acids are released from the formed aminoacyl AMP-NRPS complex, and these compounds are used to form the complex again. By repeatedly using it, the reaction product such as pyrophosphate to be measured is produced to a larger number of moles than the NRPS and / or amino acids contained in the sample, and the amount of the reaction product is measured. The method is not limited at all.

The present invention provides an amino acid quantification kit for carrying out the above-described method of the present invention, which contains the aforementioned components necessary for quantifying amino acids in a sample. The kit may appropriately contain other optional components known to those skilled in the art, such as stabilizers or buffers, to enhance the stability of reagent components such as enzymes. Although there are no particular limitations as long as it is a component that does not affect the measurement, for example, bovine serum albumin (BSA), ovalbumin, sugars, sugar alcohols, carboxyl group-containing compounds, antioxidants, surfactants, or enzymes and activity can be exemplified by amino acids without Further, as an example of the kit, the aforementioned kit for measuring pyrophosphate and hydrogen ions can be mentioned.

EXAMPLES The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited by the following examples.

(Enzyme preparation)
E. coli JM109 was transformed with a plasmid having an NRPS sequence (Pls-se-A domain, Pls-cg-A domain or Pls-cg-AT domain) derived from actinomycetes (genus Saccharopolyspora or Corynebacterium) and used as an expression strain. board. Each expression strain was cultured at 37°C in LB medium containing ampicillin at a final concentration of 200 µg/mL. IPTG was added so that the After culturing the culture at 18° C. overnight, the cells were harvested, and the resulting cells were sonicated to prepare a cell-free extract. After further centrifugation, expression of the target enzyme was confirmed by electrophoresis using a portion of the resulting supernatant. Next, the remaining supernatant was treated with a His-tag column (trade name: TALON superflow, manufactured by GE Healthcare) to remove contaminating proteins, resulting in an NRPS Pls-se-A consisting of an A domain that specifically acts on L-ornithine, Pls-cg-A of NRPS consisting of A domain that specifically acts on L-lysine and D-lysine, A domain and T domain (hereinafter referred to as AT domain) that specifically acts on L-lysine and D-lysine An NRPS consisting of Pls-cg-AT was obtained.

(Amount of pyrophosphate produced by various NRPS reactions: step (I) of the method of the present invention)
150 μL of a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 50 μM L-ornithine, and 5.0 μM Pls-se-A (derived from actinomycetes) was prepared and treated at 30° C. for 60 minutes. bottom. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After the reaction was stopped, the precipitate was removed by centrifugation to prepare a working product (product of the present invention) 1.

150 μL of a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 50 μM L-lysine or D-lysine, and 5.0 μM Pls-cg-A (derived from actinomycete) was prepared and heated to 30°C. , was processed for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare working products (products of the present invention) 2 and 3.

150 μL of a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 50 μM L-lysine or D-lysine, and 5.0 μM Pls-cg-AT (derived from actinomycete) was prepared and heated to 30°C. , was processed for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare working products (products of the present invention) 4 and 5.

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
150 μL of the prepared reaction solution of Examples 1 to 5 was mixed with 15 μL of 1 M mercaptoethanol and 60 μL of a coloring solution (2.5% ammonium molybdate/5N sulfuric acid), allowed to stand at room temperature for 20 minutes, and then the absorbance at 580 nm was measured. bottom. The amount of pyrophosphate in the reaction solution was determined from the value obtained by subtracting the absorbance value of the sample to which water was added instead of the substrate amino acid as a blank from the absorbance value of each sample. As a result, as shown in FIG. 3, both the NRPS consisting of the A domain and the AT domain had more than the number of molecules of the NRPS and more than the theoretical amount of pyrophosphate produced when each substrate amino acid was used in the enzymatic reaction. Pyrophosphate was produced. Therefore, according to the method of the present invention, it was shown that more moles of pyrophosphate are produced than the number of NRPS and amino acid molecules contained in the sample.

(Amount of Pyrophosphate Produced at Various ATP Concentrations and Enzyme Concentrations: Step (I) of the Method of the Present Invention)
Reaction solution containing 200 mM HEPES-KOH (pH 8.0), 25 mM ATP, 25 mM MnCl ₂ , 50 μM L-ornithine, 5.0 μM Pls-se-A (derived from actinomycetes), or 200 mM HEPES-KOH (pH 8.0) , 40 mM ATP, 40 mM MnCl ₂ , 50 μM L-ornithine, and 5.0 μM Pls-se-AT (derived from actinomycete) was prepared in an amount of 150 μL, and treated at 30° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare working products (products of the present invention) 6 and 7.

150 μL of a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 50 μM L-lysine, and 1.0 μM Pls-cg-A (derived from actinomycete) was prepared and treated at 30° C. for 60 minutes. bottom. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After the reaction was stopped, the precipitate was removed by centrifugation to prepare a working product (product of the present invention) 8.

Reaction solution containing 200 mM HEPES-KOH (pH 8.0), 10 mM ATP, 10 mM MnCl ₂ , 50 μM L-lysine, 5.0 μM Pls-cg-A (derived from actinomycetes), or 200 mM HEPES-KOH (pH 8.0) , 25 mM ATP, 25 mM MnCl ₂ , 50 μM L-lysine, 5.0 μM Pls-cg-A (derived from actinomycete), or 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 50 μM 150 μL of a reaction solution containing L-lysine and 5.0 μM Pls-cg-A (derived from actinomycete) was prepared and treated at 30° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and working products (products of the present invention) 9, 10 and 11 were prepared.

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
The pyrophosphates of Examples 6-11 prepared in Examples 6, 7 and 8 were measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 4, an increase in pyrophosphate was observed with an increase in ATP or enzyme concentration in various NRPSs. Moreover, more pyrophosphate was produced than the theoretical value, which is the amount of pyrophosphate produced when all the added amino acids were used in the enzymatic reaction.

(Production amount of pyrophosphate depending on the type of divalent cation: step (I) of the method of the present invention)
Reaction solution containing 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 50 μM L-ornithine, 5.0 μM Pls-se-A (derived from actinomycete), or 200 mM HEPES-KOH (pH 8.0) , 40 mM ATP, 40 mM MgCl ₂ , 50 μM L-ornithine, 5.0 μM Pls-se-A (derived from actinomycetes), or 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM CaCl ₂ , 50 μM 150 μL of a reaction solution containing L-ornithine and 5.0 μM Pls-se-A (derived from actinomycete) was prepared and treated at 30° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare working products (products of the present invention) 12, 13 and 14.

Reaction solution containing 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 50 μM L-lysine, 5.0 μM Pls-cg-A (derived from actinomycete), or 200 mM HEPES-KOH (pH 8.0) , 40 mM ATP, 40 mM MgCl ₂ , 50 μM L-lysine, 5.0 μM Pls-cg-A (derived from actinomycete), or 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM CaCl ₂ , 50 μM 150 μL of a reaction solution containing L-lysine and 5.0 μM Pls-cg-A (derived from actinomycete) was prepared and treated at 30° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare working products (products of the present invention) 15, 16 and 17.

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
Pyrophosphate of Examples 12 to 17 prepared in Examples 10 and 11 was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 5, under the conditions using various divalent cations, more pyrophosphate than the theoretical value, which is the amount of pyrophosphate produced when the substrate amino acid is used in the enzymatic reaction, was produced. rice field. Also, Mn was shown to be the divalent cation of choice, at least for the NRPS used in the above examples.

(Amount of Pyrophosphate Produced by Difference in Divalent Cation Concentration: Step (I) of the Method of the Present Invention)
Reaction solution containing 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MgCl ₂ , 50 μM L-ornithine, 5.0 μM Pls-se-A (derived from actinomycete), or 200 mM HEPES-KOH (pH 8.0) , 40 mM ATP, 120 mM MgCl ₂ , 50 μM L-ornithine, 5.0 μM Pls-se-A (derived from actinomycetes), or 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 200 mM MgCl ₂ , 50 μM Reaction solution containing L-ornithine, 5.0 μM Pls-se-A (derived from actinomycetes), or 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 400 mM MgCl ₂ , 50 μM L-ornithine, 5.0 μM Pls- 150 μL of a reaction solution containing se-A (derived from actinomycete) was prepared and treated at 30° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare working products (products of the present invention) 18 to 21.

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
The pyrophosphate of Examples 18-21 prepared in Example 13 was determined by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 6, it was shown that the amount of pyrophosphate produced by the enzymatic reaction varied depending on the divalent cation concentration.

(Pyrophosphate production amount by reaction pH)
200 mM MES (pH 6.0), or 200 mM HEPES-KOH (pH 7.0), or 200 mM HEPES-KOH (pH 7.5), or 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 50 μM 150 μL of a reaction solution containing L-ornithine and 5.0 μM Pls-se-A (derived from actinomycete) was prepared and treated at 30° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and pyrophosphate in the supernatant was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 7, the more alkaline the pH, the more the amount of pyrophosphate produced, and good pyrophosphate production was observed at pH 8.0.

(Pyrophosphate production amount by reaction pH)
200 mM MES (pH 6.0), or 200 mM HEPES-KOH (pH 7.0), or 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 50 μM L-lysine, 2.5 μM Pls-cg- 150 μL of a reaction solution containing A (derived from actinomycete) was prepared and treated at 30° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and pyrophosphate in the supernatant was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 7, the more alkaline the pH, the more the amount of pyrophosphate produced, and good pyrophosphate production was observed at pH 8.0.

(Amount of pyrophosphate produced by reaction temperature)
Prepare 150 μL of a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 50 μM L-ornithine, 5.0 μM Pls-se-A (derived from actinomycete), 10, or 20, or Treated at 25, or 30, or 35, or 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and pyrophosphate in the supernatant was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 8, good pyrophosphate production was observed over a wide range of 10 to 40.degree.

(Comparison of the amount of pyrophosphate produced in the present invention and the NRPS reaction described in known literature: Step (I) of the method of the present invention)
[Comparative Example 1]
According to the reaction conditions described in Non-Patent Document 7, 0.9 μM Pls-cg-A (derived from actinomycetes), 50 μM L-lysine, 5 mM ATP, 5 mM MgCl ₂ , 50 mM Tris-HCl (pH 8.0), 32 mM hydroxylamine. A reaction solution containing 300 μL was prepared, and an enzymatic reaction was performed at 30° C. for 60 minutes. After the enzymatic reaction, 60 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare a comparative product 1.

[Comparative Example 2]
According to the reaction conditions described in Non-Patent Document 7, 0.8 μM Pls-cg-AT (derived from actinomycetes), 50 μM L-lysine, 5 mM ATP, 5 mM MgCl ₂ , 50 mM Tris-HCl (pH 8.0), 32 mM hydroxylamine. A reaction solution containing 300 μL was prepared, and an enzymatic reaction was performed at 30° C. for 60 minutes. After the enzymatic reaction, 60 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare a comparative product 2.

300 μL of a reaction solution containing 5.0 μM Pls-cg-A (derived from actinomycetes), 50 μM L-lysine, 40 mM ATP, 40 mM MnCl ₂ , 200 mM HEPES-KOH (pH 8.0) was prepared and incubated with the enzyme at 30° C. for 60 minutes. reacted. After the enzymatic reaction, 60 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After the reaction was stopped, the precipitate was removed by centrifugation to prepare Working Product 22.

300 μL of a reaction solution containing 5.0 μM Pls-cg-AT (derived from actinomycetes), 50 μM L-lysine, 40 mM ATP, 40 mM MnCl ₂ , 200 mM HEPES-KOH (pH 8.0) was prepared and incubated with the enzyme at 30° C. for 60 minutes. reacted. After the enzymatic reaction, 60 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After the reaction was stopped, the precipitate was removed by centrifugation to prepare Working Product 23.

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
The pyrophosphates of Comparative Products 1 and 2 and Examples 22 and 23 obtained in Comparative Examples 1 and 2 and Examples 16 and 17 were measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 9, in the method of the present invention, more pyrophosphate was produced than the theoretical value, which is the amount of pyrophosphate expected to be produced when all the added amino acids were used in the reaction. rice field. On the other hand, the amount of pyrophosphate produced by the comparative product was below the theoretical value.

(Amino acid calibration curve in pyrophosphate measurement by molybdenum blue method)
Reaction containing 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 0, or 30, or 50, or 70, or 100 μM L-ornithine, 5.0 μM Pls-se-A (from actinomycete) 150 μL of solution was prepared and reacted at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and pyrophosphate in the supernatant was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 10, more pyrophosphate was produced than the theoretical amount of pyrophosphate that would be produced if all the added amino acids were used in the reaction. Moreover, a correlation (R=0.99) was observed between the amino acid concentration and the amount of pyrophosphate in the amino acid concentration range of 0 to 100 μM, indicating that L-ornithine can be quantified.

Reaction containing 200 mM HEPES-KOH (pH 8.0), 40 mM ATP, 40 mM MnCl ₂ , 0, or 30, or 50, or 70, or 100 μM L-lysine, 5.0 μM Pls-cg-A (from actinomycete) 150 μL of solution was prepared and reacted at 30° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and pyrophosphate in the supernatant was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 10, more pyrophosphate was produced than the theoretical amount of pyrophosphate that would be produced if all the added amino acids were used in the reaction. Moreover, a correlation (R=0.97) was observed between the amino acid concentration and the amount of pyrophosphate in the amino acid concentration range of 0 to 100 μM, indicating that L-lysine can be quantified.

From the above results, it was shown that in the NRPS reaction in the method of the present invention, the pyrophosphate produced can be amplified by repeatedly using NRPS and amino acids in the reaction. As shown in Examples 2 to 5, any of the various amino acids L-ornithine, L-lysine and D-lysine produce more pyrophosphate produced in the reaction than the number of moles of NRPS and amino acids. was made. As shown in Examples 6 to 9, the amount of pyrophosphate produced varies depending on ATP and enzyme concentrations, and the type and concentration of divalent cations, reaction temperature, and reaction pH, as shown in Examples 10 to 17. I found out to do. In addition, as shown in Comparative Examples 1 and 2 and Examples 18, 19, and 20, in the conventional NRPS reaction, the amount of pyrophosphate produced was less than the number of amino acid molecules, but in the method of the present invention, It was found that more pyrophosphate was produced than the number of amino acid molecules. Furthermore, as shown in Examples 21 and 22, it was found that pyrophosphate increases linearly depending on the amino acid concentration, that is, there is a correlation between the amino acid concentration and the amount of pyrophosphate. It was confirmed that it was possible to quantify various amino acids from the produced pyrophosphate using the molybdenum blue method, which is a simple method.

In the conventional amino acid quantification method using AARS, there are 20 types of AARS specific for proteinogenic amino acids (20 types of L-type amino acids that make up proteins), but other than 20 types of amino acids such as ornithine, Since there is no AARS that does this, there are restrictions on the amino acids that can be measured. On the other hand, NRPS acts specifically on the above 20 kinds of amino acids, and also isomers of D-type amino acids, modified amino acids such as methylation, acylation, and glycosylation, and non-amino acids such as ornithine. Since it also acts specifically on proteinogenic amino acids, it enables the quantification of various amino acids. In addition, in the method of the present invention, by releasing NRPS and amino acids from the formed aminoacyl AMP-NRPS conjugate and repeatedly using them again to form the aminoacyl AMP-NRPS conjugate, only a small amount of amino acids is present in the sample. Even if it is not contained, a large amount of pyrophosphate and hydrogen ions can be produced, so high-sensitivity analysis by fluorescence method or the like using multistep enzymatic reaction is unnecessary. Therefore, the present invention provides a method and a kit for amino acid quantification that are specific, convenient, and highly sensitive for quantifying proteinogenic amino acids, D-amino acids, modified amino acids, and non-proteinogenic amino acids such as ornithine using NRPS. became possible.

Specifically, each NRPS prepared in Example 1 was obtained as follows.
For NRPS that is expected to catalyze the activation of the target amino acid, a domain search was performed on the deduced amino acid sequence by Pfam search (http://pfam.xfam.org/search#tabview=tab1). Based on the results, highly conserved regions in each NRPS domain (A domain, T domain, etc.) necessary for peptide bond formation were deduced, and the sequences were determined in *1 and *2 below.
*1. Sequence highly conserved in the A domain AMP-binding domain (underlined)
AMP-binding enzyme C-terminal domain (double line)
*2. A PP-binding domain centered on the sequence LGGxxSLxA motif (4'-phosphopantetheine attachment site) highly conserved in the T domain (inside the square frame).

アミノ酸配列（実際に活性測定に用いた組換え酵素）：
［配列番号２］

The amino acid sequences of each NRPS are as follows.
(1) Pls-cg-A-domain
Strain name: Corynebacterium glutamicum NBRC12169
Amino acid sequence (region cloned into E. coli expression plasmid):
[SEQ ID NO: 1]

Amino acid sequence (recombinant enzyme actually used for activity measurement):
[SEQ ID NO: 2]

アミノ酸配列（実際に活性測定に用いた組換え酵素）：
［配列番号４］

(2) Pls-cg-AT-domain
Strain name: Corynebacterium glutamicum NBRC12169
Amino acid sequence (region cloned into E. coli expression plasmid):
[SEQ ID NO: 3]

Amino acid sequence (recombinant enzyme actually used for activity measurement):
[SEQ ID NO: 4]

アミノ酸配列（実際に活性測定に用いた組換え酵素）：
［配列番号６］

(3) Pls-se-A-domain
Strain name: Saccharopolyspora erythraea NBRC13426
Amino acid sequence (region cloned into E. coli expression plasmid):
[SEQ ID NO: 5]

Amino acid sequence (recombinant enzyme actually used for activity measurement):
[SEQ ID NO: 6]

Claims (7)

Step (I) comprising the following steps:
(Step I-1) In the presence of divalent cations, an amino acid (AA) in a sample, a non-ribosomal peptide synthetase (NRPS) corresponding to the AA, and adenosine triphosphate (ATP) are reacted. , a reaction (reaction 1) to form a conjugate consisting of aminoacyl adenylic acid (aminoacyl AMP) and NRPS (aminoacyl AMP-NRPS conjugate);
(Step I-2) A step including a reaction (Reaction 2) in which an aminoacyl AMP-NRPS complex formed in Reaction 1 and/or Reaction 3 is reacted with an amino acid regenerating reagent to liberate NRPS and AA from the complex. ;
(Step I-3) a step comprising a reaction (reaction 3) to form an aminoacyl AMP-NRPS conjugate reaction by recycling the AA and/or NRPS liberated in reaction 2 in reaction 1;
(Step I-4) repeating steps I-2 and I-3, and
step (II) comprising measuring the amount of the reaction product generated in step (I) and determining the amount of the amino acid based on the measured amount of the reaction product;
and wherein said NRPS comprises at least an A domain. 2. The method for quantifying amino acids according to claim 1, wherein the NRPS is composed of an A domain and/or composed of an A domain and a T domain. 3. The method for quantifying amino acids according to claim 1 or 2, wherein the amino acid regeneration reagent used in step (I) is a nucleotide and/or an alkaline compound. 4. The method for quantifying amino acids according to any one of claims 1 to 3, wherein the amount of the reaction product produced in step (I) is measured by measuring absorbance change by an absorbance method. 5. The method for quantifying amino acids according to any one of claims 1 to 4, wherein at least one of pyrophosphate and hydrogen ions is measured as the reaction product generated in step (I). The method for quantifying amino acids according to any one of claims 1 to 5, wherein the number of moles of the reaction product produced in step (I) is greater than the number of moles of NRPS and/or amino acids in the sample. An amino acid quantification kit for carrying out the amino acid quantification method according to any one of claims 1 to 6, comprising ATP, a nucleotide and an NRPS corresponding to said amino acid.

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