JPWO2019065211A1 - Amino acid quantification method and amino acid quantification kit - Google Patents

Amino acid quantification method and amino acid quantification kit Download PDF

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JPWO2019065211A1
JPWO2019065211A1 JP2019544537A JP2019544537A JPWO2019065211A1 JP WO2019065211 A1 JPWO2019065211 A1 JP WO2019065211A1 JP 2019544537 A JP2019544537 A JP 2019544537A JP 2019544537 A JP2019544537 A JP 2019544537A JP WO2019065211 A1 JPWO2019065211 A1 JP WO2019065211A1
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朋子 中柄
朋子 中柄
青木　秀之
秀之 青木
幹子 喜田
幹子 喜田
健太 山田
健太 山田
吉十 濱野
吉十 濱野
千登勢 丸山
千登勢 丸山
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Abstract

【課題】測定対象のタンパク質性アミノ酸（タンパク質を構成する２０種のＬ型のアミノ酸）、また、Ｄ型アミノ酸、修飾アミノ酸、及びオルニチン等の非タンパク質性アミノ酸を特異的且つ簡便、高感度に定量する方法及びアミノ酸定量用キットを提供すること。【解決手段】非リボソーム型ペプチド合成酵素（ＮＲＰＳ）を用いて、試料中のアミノ酸量を定量する方法に於いて、一度形成させたアミノアシルＡＭＰ-ＮＲＰＳ複合体からＮＲＰＳ及びアミノ酸を遊離させ、それらを再度、アミノアシルＡＭＰ-ＮＲＰＳ複合体の形成に利用することによって、最終的に、測定対象であるピロリン酸等の反応産生物を、試料中に含まれるＮＲＰＳ及び／又はアミノ酸より多くのモル数まで産生させることを特徴とする、前記アミノ酸定量方法、及び、該方法を実施するためのアミノ酸定量用キット。【選択図】図１PROBLEM TO BE SOLVED: To specifically, easily and highly sensitively quantify proteinaceous amino acids to be measured (20 kinds of L-type amino acids constituting a protein), D-type amino acids, modified amino acids, and non-proteinaceous amino acids such as ornithine. And provide a kit for amino acid quantification. SOLUTION: In a method of quantifying the amount of amino acids in a sample using a nonribosomal peptide synthase (NRPS), NRPS and amino acids are released from an aminoacyl AMP-NRPS complex once formed, and they are released. By using it again for the formation of the aminoacyl AMP-NRPS complex, the reaction product such as pyrophosphate to be measured is finally produced up to the number of moles larger than the NRPS and / or amino acid contained in the sample. The amino acid quantification method, and an amino acid quantification kit for carrying out the method. [Selection diagram] Fig. 1

Description

本発明は、アミノ酸定量方法及びアミノ酸定量用キット等に関する。 The present invention relates to an amino acid quantification method, an amino acid quantification kit, and the like.

アミノ酸は、生体内のタンパク質の構成成分として重要な役割を担っている。アミノ酸の機能性に関し多くの研究がなされ、アミノ酸は、医薬品、加工食品、健康食品など様々な産業で使用されている。例えば、食品中の遊離アミノ酸は、味、加熱後の香り、保存性、摂取後の生体調節機能等に関係しており、食品科学や栄養科学分野における重要な要素として注目されている。また、近年、疾病により血液中のアミノ酸濃度が変化することが見いだされ、血液中のアミノ酸濃度を測定することで、肺がん、胃がん、大腸がんなどのがん診断を可能とするバイオマーカーとしても活用されている。その為、アミノ酸定量技術は、アミノ酸を使用する製品開発、品質管理、診断などの様々な分野で必要不可欠な技術となっている。 Amino acids play an important role as a constituent of proteins in the body. Much research has been done 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 foods are related to taste, aroma after heating, storage stability, bioregulatory function after ingestion, etc., and are attracting attention as important elements in the fields of food science and nutrition science. In recent years, it has been found that the amino acid concentration in blood changes due to diseases, and by measuring the amino acid concentration in blood, it can be used as a biomarker that enables cancer diagnosis such as lung cancer, stomach cancer, and colon cancer. It is being utilized. Therefore, amino acid quantification technology has become an indispensable technology in various fields such as product development using amino acids, quality control, and diagnosis.

アミノ酸定量技術として、アミノ酸を液体クロマトグラフィーで分離し、ニンヒドリンやオルトフタルアルデヒドによる呈色反応で検出する方法が知られている（非特許文献１）。しかし、該方法は、１検体の分析時間が２時間程度必要なため分析時間がかかり、多数の検体を測定するには不向きであるという問題点があった。 As an amino acid quantification technique, a method of separating amino acids by liquid chromatography and detecting them by a color reaction with ninhydrin or orthophthalaldehyde is known (Non-Patent Document 1). However, this method has a problem that it takes about 2 hours to analyze one sample, so that it takes a long time to analyze and is not suitable for measuring a large number of samples.

別のアミノ酸定量技術として、アミノ酸に作用する酵素を利用するアミノ酸の測定方法が知られている（特許文献１）。しかし、該方法は目的基質とするアミノ酸に対する選択性が低く、目的以外のアミノ酸にも反応する問題点があった。 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 a problem that it has low selectivity for an amino acid as a target substrate and reacts with an amino acid other than the target.

アミノアシルｔＲＮＡ合成酵素（ＡＡＲＳ）は、生体内のタンパク質合成に係る酵素であり、２０種類のアミノ酸に対し特異的な２０種類のＡＡＲＳが存在する。その為、標的とするアミノ酸に対する選択性が極めて高い酵素であるとされ、ＡＡＲＳが関与する反応（ＡＡＲＳ反応）を利用したアミノ酸定量技術がこれまで開発されてきた。例えば、ＡＡＲＳを用いたアミノ酸の分析方法が特許文献２に記載されている。該方法では、ＡＡＲＳが触媒として機能してＡＴＰ、アミノ酸が1分子ずつ反応することでアミノアシルＡＭＰとピロリン酸が産生され、産生するピロリン酸を指標とすることによりアミノ酸を分析する。しかしながら、該方法ではＡＡＲＳが1分子のアミノ酸とＡＴＰと反応し、1分子のアミノアシルＡＭＰを産生するため、試料中のアミノ酸と同量かそれ以下のピロリン酸しか産生されない（非特許文献２、３、４）。 Aminoacyl-tRNA synthetase (AARS) is an enzyme involved in protein synthesis in vivo, and there are 20 types of AARS specific for 20 types of amino acids. Therefore, it is considered to be an enzyme having extremely high selectivity for the target amino acid, and an amino acid quantification technique using a reaction involving AARS (AARS reaction) has been developed so far. For example, a method for analyzing amino acids using AARS is described in Patent Document 2. In this method, AARS functions as a catalyst and ATP and amino acids react one molecule at a time to produce aminoacyl AMP and pyrophosphoric acid, and the amino acids are analyzed by using the produced pyrophosphate as an index. However, in this method, AARS reacts with one molecule of amino acid and ATP to produce one molecule of aminoacyl AMP, so that only the same amount or less of pyrophosphate as the amino acid in the sample is produced (Non-Patent Documents 2 and 3). 4).

更に、ＡＡＲＳ反応に基づくアミノ酸の定量方法が上記の特許文献３に記載されている。即ち、前述の通りアミノアシルＡＭＰは、通常、ＡＡＲＳに強く結合してアミノアシルＡＭＰ-ＡＡＲＳ複合体を形成している。そこで、この方法では、アミノアシルＡＭＰ-ＡＡＲＳ複合体分解試薬としてヒドロキシルアミンなどのアミン類（求核剤）を添加することで、ＡＡＲＳを再び反応可能な状態とし、その結果、少量のＡＡＲＳでアミノ酸を定量することを特徴とする。しかしながら、特許文献３に記載の方法は、該複合体分解試薬が複合体のアミノ酸と反応して化合物が産生される（例えば、特許文献３の段落番号００３７に示されるように、該複合体分解試薬としてヒドロキシルアミンを使用した場合は、「アミノ酸ヒドロキサム酸」が産生される）ため、アミノ酸は再利用することが出来ず、その結果、試料中のアミノ酸と同等量の産生物であるピロリン酸しか得られない（特許文献３の段落番号００２３）。 Further, a method for quantifying amino acids based on the AARS reaction is described in Patent Document 3 above. That is, as described above, aminoacyl AMP usually binds strongly to AARS to form an aminoacyl AMP-AARS complex. Therefore, in this method, amines such as hydroxylamine (nucleophile) are added as an aminoacyl AMP-AARS complex decomposition reagent to make AARS reactive again, and as a result, amino acids are added with a small amount of AARS. It is characterized by quantification. However, in the method described in Patent Document 3, the complex decomposition reagent reacts with the amino acid of the complex to produce a compound (for example, as shown in paragraph No. 0037 of Patent Document 3, the complex decomposition is performed. When hydroxylamine is used as a reagent, "amino acid hydroxamic acid" is produced), so the amino acid cannot be reused, and as a result, only pyrophosphate, which is the same amount of product as the amino acid in the sample, is produced. Not obtained (paragraph number 0023 of Patent Document 3).

このように、ＡＡＲＳが関与する該方法は、ピロリン酸産生量が少ないため、高感度分析を行う問題点があった。さらにＡＡＲＳは、２０種類のアミノ酸に対する特異的な２０種類のＡＡＲＳが存在するが、オルニチンなどの２０種類のアミノ酸以外に反応するＡＡＲＳは存在しない課題もある。 As described above, the method involving AARS has a problem of performing high-sensitivity analysis because the amount of pyrophosphate produced is small. Further, there is a problem that there are 20 kinds of AARS specific to 20 kinds of amino acids, but there is no AARS that reacts with other than 20 kinds of amino acids such as ornithine.

非リボソーム型ペプチド合成酵素（ＮＲＰＳ）は、複合ペプチド合成に関与する酵素であり、抗生物質などの合成に利用されている。ＮＲＰＳは、タンパク質性アミノ酸（タンパク質を構成する２０種のＬ型のアミノ酸）に作用すると共に、異性体であるＤ型のアミノ酸、さらにはメチル化、アシル化、グリコシル化などの修飾アミノ酸及びオルニチン等の非タンパク質性アミノ酸（タンパク質の構成単位ではないアミノ酸）にも作用し、更に、選択性（特異性）が極めて高い酵素であるとされている。ＮＲＰＳは、アデニレーション・ドメイン（Ａドメイン）、チオレーション・ドメイン（Ｔドメイン）、コンデンセーション・ドメイン（Ｃドメイン）の３つのドメインから構成される。ＮＲＰＳ反応は、図１に示す通りで、Ａドメインでは、ＮＲＰＳにアデノシン三リン酸（ＡＴＰ）、アミノ酸（ＡＡ）が１分子ずつ作用することで、アミノアシルアデニル酸（アミノアシルＡＭＰ）とピロリン酸（ＰＰｉ）が産生される。次いでＴドメインでは、アミノアシルＡＭＰとＴドメインのアミノアシル４’ホスホパンテテイン基（４’ＰＰ）が反応し、アミノアシル-Ｔドメイン複合体を形成する。続いてＣドメインでは、アミノアシル-Ｔドメイン複合体に結合しているアミノ酸同士を結合させるペプチド縮合反応を起こさせることで、ペプチドが産生する。ＮＲＰＳは、このような反応を繰り返すことでアミノ酸が３〜１５量体の直線状或いは環状に連なったペプチドを合成する（非特許文献５、非特許文献６）。 The nonribosomal peptide synthase (NRPS) is an enzyme involved in complex peptide synthesis and is used for the synthesis of antibiotics and the like. NRPS acts on proteinaceous amino acids (20 kinds of L-type amino acids that make up proteins), and is an isomer D-type amino acid, as well as modified amino acids such as methylation, acylation, and glycosylation, ornithine, etc. It is said to be an enzyme that also acts on non-protein amino acids (amino acids that are not constituent units of proteins) and has extremely high selectivity (specificity). The NRPS is composed of three domains: an adenation 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, adenosine triphosphate (ATP) and amino acid (AA) act on NRPS one molecule at a time to form aminoacyl-adenylic acid (aminoacyl AMP) and pyrophosphate (PPi). ) Is produced. Then, in the T domain, the aminoacyl AMP reacts with the aminoacyl 4'phosphopanthetein group (4'PP) in the T domain to form an aminoacyl-T domain complex. Subsequently, in the C domain, peptides are produced by causing a peptide condensation reaction that binds amino acids bound to the aminoacyl-T domain complex. By repeating such a reaction, NRPS synthesizes a peptide in which amino acids are linearly or cyclically linked in 3 to 15 dimers (Non-Patent Documents 5 and 6).

ＮＲＰＳに関する公知文献（特許文献４、５、非特許文献５、６）に記載された技術は、Ａ、Ｔ及びＣドメインから成るＮＲＰＳによるペプチド合成に関するものであって、Ａドメイン、又は、Ａドメイン及びＴドメインから成るＮＲＰＳによるアミノ酸の定量に関する技術的課題を解決することを目的とするものではない。また、ＮＲＰＳの公知文献（非特許文献７）では、バイオポリマー合成のために使用するＮＲＰＳの活性測定法の記載があるが、１分子のＮＲＰＳに対し１分子のアミノ酸とＡＴＰが反応し、アミノアシルＡＭＰ-Ａドメイン複合体を形成するため、試料中のＮＲＰＳ濃度までしか反応しないことから、アミノアシルＡＭＰ-ＮＲＰＳ複合体分解試薬としてヒドロキシルアミン（アミン類の求核剤）を添加することで、ＮＲＰＳを再び反応可能な状態とし、ＮＲＰＳの活性確認をしている。しかしながら、本方法は、該複合体分解試薬が複合体のアミノ酸と反応して化合物が産生されるため、アミノ酸は再利用することが出来ず、その結果、試料中のアミノ酸と同等量の産生物であるピロリン酸しか生成されない。これら公知文献には、ＮＲＰＳ反応を活用したアミノ酸の定量、及び、アミノ酸の定量にＡドメイン、又は、Ａドメイン及びＴドメインから成るＮＲＰＳを用いることについては何ら言及されておらず、更に、アミノ酸とＮＲＰＳとの反応におけるアミノ酸及びＮＲＰＳの再利用に関しても一切触れられていない。 The techniques described in publicly known documents relating to NRPS (Patent Documents 4 and 5, Non-Patent Documents 5 and 6) relate to peptide synthesis by NRPS consisting of A, T and C domains, and are A domain or A domain. It is not intended to solve the technical problems related to the quantification of amino acids by NRPS consisting of T domain and T domain. Further, in a known document of NRPS (Non-Patent Document 7), there is a description of a method for measuring the activity of NRPS used for biopolymer synthesis, but one molecule of amino acid reacts with ATP to one molecule of NRPS, and aminoacyl Since it reacts only up to the NRPS concentration in the sample to form an AMP-A domain complex, NRPS can be obtained by adding hydroxylamine (a nucleophilic agent for amino acids) as an aminoacyl AMP-NRPS complex decomposition reagent. The reaction is made ready again, and the activity of NRPS is confirmed. However, in this method, since the complex decomposition reagent reacts with the amino acid of the complex to produce a compound, the amino acid cannot be reused, and as a result, the same amount of product as the amino acid in the sample is produced. Only pyrophosphate is produced. These publicly known documents do not mention the quantification of amino acids utilizing the NRPS reaction and the use of NRPS consisting of A domain or A domain and T domain for quantification of amino acids. No mention is made of the reuse of amino acids and NRPS in the reaction with NRPS.

特開２０１３−１４６２６４号公報Japanese Unexamined Patent Publication No. 2013-146264 特開２０１１−５０３５７号公報Japanese Unexamined Patent Publication No. 2011-50357 特許５３０５２０８号明細書Patent No. 5305208 ＷＯ２００６／００１３８２WO2006 / 001382 特許５３６７２７２号明細書Japanese Patent No. 5376272

“化学と生物”、（日本）、２０１５年、Ｖｏｌ.５３、ｐ．１９２−１９７"Chemistry and Biology", (Japan), 2015, Vol.53, p. 192-197 “広島市立大学情報科学研究科創造科学専攻 釘宮章光”[online]、 広島市立大ホームページ、［2016/01/13検索］、インターネット（URL:http://rsw.office.hiroshima-cu.ac.jp/Profiles/2/000129/profile.html）"Hiroshima City University Graduate School of Information Science, Department of Creative Science, Akimitsu Nagimiya" [online], Hiroshima City University website, [2016/01/13 search], Internet (URL: http://rsw.office.hiroshima-cu.ac. jp / Profiles / 2/000129/profile.html) Ａｎａｌｙｔｉｃａｌ Ｂｉｏｃｈｅｍ., ４４３, ２２−２６, ２０１３Analytical Biochem., 443, 22-26, 2013 Ａｐｐl. Ｂｉｏｃｈｅｍ. Ｂｉｏｔｅｃｈｎｏl., １７４, ２５２７−２５３６, ２０１４Apple. Biochem. Biotechnol., 174, 2527-2536, 2014 “社団法人日本化学会 ＮＥＷＳ ＬＥＴＴＥＲ”、（日本）、２０１５年、Ｖｏｌ.２９、ｐ．３−６"The Chemical Society of Japan NEWS LETTER", (Japan), 2015, Vol.29, p. 3-6 “化学と生物”、（日本）、２００６年、Ｖｏｌ.４４、ｐ．８５−９２"Chemistry and Biology", (Japan), 2006, Vol.44, p. 85-92 Ａｎａｌｙｔｉｃａｌ Ｓｃｉｅｎｃｅｓ Ｊａｎｕａｒｙ, ３０, １７−２４, ２０１４Analytical Sciences January, 30, 17-24, 2014

本発明は、上記のアミノ酸定量法に関する従来技術に於ける様々な問題点を解決し、測定対象のタンパク質性アミノ酸（タンパク質を構成する２０種のＬ型のアミノ酸）、また、Ｄ型アミノ酸、修飾アミノ酸及びオルニチン等の非タンパク質性アミノ酸を特異的且つ簡便、高感度に定量する方法及びアミノ酸定量用キットを提供することを目的とする。 The present invention solves various problems in the prior art regarding the above-mentioned amino acid quantification method, and measures proteinaceous amino acids (20 kinds of L-type amino acids constituting the protein), D-type amino acids, and modifications. It is an object of the present invention to provide a method for quantifying non-protein amino acids such as amino acids and ornithine in a specific, simple and highly sensitive manner, and a kit for quantifying amino acids.

ＮＲＰＳを用いて試料中のアミノ酸量を定量する方法に於いて、従来のＡ、Ｔ及びＣドメインから成るＮＲＰＳ反応では、ＮＲＰＳに対しアミノ酸とＡＴＰが反応し、ペプチドを産生するため、試料中のアミノ酸を再利用できないと共に、アミノ酸と同量かそれ以下のピロリン酸しか産生されない、と考えられる。また、Ａ及びＴドメインから成るＮＲＰＳ反応では、１分子のＮＲＰＳに対し１分子のアミノ酸とＡＴＰが反応するが、アミノアシル-Ｔドメイン複合体を形成するため、試料中のアミノ酸を再利用できないと共に、試料中のＮＲＰＳと同量かそれ以下のピロリン酸しか産生されない、と考えられる。さらにＡドメインから成るＮＲＰＳ反応では、１分子のＮＲＰＳに対し１分子のアミノ酸とＡＴＰが反応するが、アミノアシルＡＭＰ-Ａドメイン複合体を形成するため、試料中のＮＲＰＳと同量かそれ以下のピロリン酸しか産生されない、と考えられる。そこで発明者らは、種々の検討を行った結果、図２に示すように、一度形成させたアミノアシルＡＭＰ-ＮＲＰＳ複合体からＮＲＰＳ及びアミノ酸（ＡＡ）を遊離させ、それらを再度、アミノアシルＡＭＰ-ＮＲＰＳ複合体の形成に利用することによって、最終的に、測定対象であるピロリン酸等の反応産生物が、試料中に含まれるＮＲＰＳ及び／又はアミノ酸より多くのモル数まで産生され得ることを見出し、本発明を完成させた。 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 considered that amino acids cannot be reused and only pyrophosphates equal to or less than amino acids are produced. Further, in the NRPS reaction consisting of A and T domains, one molecule of amino acid reacts with ATP for one molecule of NRPS, but since an aminoacyl-Tdomain complex is formed, the amino acid in the sample cannot be reused and the amino acid in the sample cannot be reused. It is considered that only pyrophosphate equal to or less than the NRPS in the sample is produced. Furthermore, in the NRPS reaction consisting of the A domain, one molecule of amino acid and ATP react with one molecule of NRPS, but since an aminoacyl AMP-A domain complex is formed, the amount of pyrolin is equal to or less than that of the NRPS in the sample. It is believed that only acid is produced. Therefore, as a result of various studies, the inventors released NRPS and an amino acid (AA) from the aminoacyl AMP-NRPS complex once formed, and re-released them as aminoacyl AMP-NRPS. By utilizing it for the formation of the complex, it was finally found that the reaction product such as pyrophosphate to be measured can be produced up to the number of moles larger than the NRPS and / or amino acid contained in the sample. The present invention has been completed.

本発明は、以下の［１］〜［６］の態様に関する。
［１］以下の各工程を含む工程（Ｉ）：
（工程Ｉ−１）二価陽イオンの存在下、試料中のアミノ酸（ＡＡ）、該ＡＡに対応する非リボソーム型ペプチド合成酵素（ＮＲＰＳ）、及び、アデノシン三リン酸（ＡＴＰ）を反応させて、アミノアシルアデニル酸（アミノアシルＡＭＰ）とＮＲＰＳから成る複合体（アミノアシルＡＭＰ-ＮＲＰＳ複合体）を形成させる反応（反応１）を含む工程；
（工程Ｉ−２）反応１及び／又は反応３で形成されたアミノアシルＡＭＰ-ＮＲＰＳ複合体にアミノ酸再生試薬が作用して、該複合体からＮＲＰＳ及びＡＡが遊離する反応（反応２）を含む工程；
（工程Ｉ−３）反応２で遊離されたＡＡ及び/又はＮＲＰＳを反応１において再利用することによってアミノアシルＡＭＰ-ＮＲＰＳ複合体反応を形成させる反応（反応３）を含む工程；及び、
（工程Ｉ−４）工程Ｉ−２及び工程Ｉ−３を繰り返す工程、並びに、
工程（Ｉ）で生じた反応産生物の量を測定し、該反応産生物の測定量に基づきアミノ酸の量を決定することを含む工程（ＩＩ）、
を含み、前記ＮＲＰＳは少なくともＡドメインを含む、試料中のアミノ酸定量方法。
［２］ＮＲＰＳがＡドメインから構成されるもの、並びに/又は、Ａドメイン及びＴドメインから構成されるものである、［１］に記載のアミノ酸定量方法。
［３］工程（Ｉ）で用いるアミノ酸再生試薬が、ヌクレオチド及び／又はアルカリ性化合物であることを特徴とする、［１］に記載のアミノ酸定量方法。
［４］吸光度法により吸光度変化を測定することによって、工程（Ｉ）で生じた反応産生物の量を測定する、［１］〜［３］の何れか一項に記載のアミノ酸定量方法。
［５］工程（Ｉ）で生じる反応産生物として、ピロリン酸又は水素イオンの少なくとも何れか１つを測定する、［１］〜［４］の何れか一項に記載のアミノ酸定量方法。
［６］工程（Ｉ）で生じた反応産生物のモル数が試料中のＮＲＰＳ及び／又はアミノ酸のモル数より多いことを特徴とする、［１］〜［５］のいずれか一項に記載のアミノ酸定量方法。
［７］［１］〜［６］に記載のアミノ酸定量法を実施するためのアミノ酸定量用キットであって、ＡＴＰ、ヌクレオチド及び該アミノ酸に対応するＮＲＰＳを含む、アミノ酸定量用キット。The present invention relates to the following aspects [1] to [6].
[1] Step (I) including each of the following steps:
(Step I-1) In the presence of divalent cations, the amino acid (AA) in the sample, the nonribosomal peptide synthase (NRPS) corresponding to the AA, and adenosine triphosphate (ATP) are reacted. , A step comprising a reaction (reaction 1) to form a complex (aminoacyl AMP-NRPS complex) consisting of aminoacyl-adenylic acid (aminoacyl AMP) and NRPS;
(Step I-2) A step including a reaction (Reaction 2) in which an amino acid regeneration reagent acts on the aminoacyl AMP-NRPS complex formed in Reaction 1 and / or Reaction 3 to release NRPS and AA from the complex. ;
(Step I-3) A step including a reaction (reaction 3) in which the AA and / or NRPS released in the reaction 2 is reused in the reaction 1 to form an aminoacyl AMP-NRPS complex reaction;
(Step I-4) A step of repeating steps I-2 and I-3, and
Step (II), which comprises measuring the amount of reaction product produced in step (I) and determining the amount of amino acid based on the measured amount of the reaction product.
A method for quantifying amino acids in a sample, wherein the NRPS contains at least the A domain.
[2] The amino acid quantification method according to [1], wherein the NRPS is composed of an A domain and / or an A domain and a T domain.
[3] The amino acid quantification method according to [1], wherein the amino acid regeneration reagent used in step (I) is a nucleotide and / or an alkaline compound.
[4] The amino acid quantification method according to any one of [1] to [3], wherein the amount of the reaction product produced in the step (I) is measured by measuring the change in absorbance by the absorbance method.
[5] The amino acid quantification method according to any one of [1] to [4], wherein at least one of pyrophosphate or hydrogen ion is measured as the reaction product produced in the step (I).
[6] The item according to any one of [1] to [5], wherein the number of moles of the reaction product produced in the step (I) is larger than the number of moles of the NRPS and / or amino acid in the sample. Amino acid quantification method.
[7] An amino acid quantification kit for carrying out the amino acid quantification method according to [1] to [6], which comprises ATP, nucleotides and NRPS corresponding to the amino acid.

本発明に係るアミノ酸定量方法に於いては、タンパク質性アミノ酸（タンパク質を構成する２０種のＬ型のアミノ酸）が特異的に測定できると共に、異性体であるＤ型のアミノ酸、さらにはメチル化、アシル化、グリコシル化などの修飾アミノ酸及びオルニチン等の非タンパク質性アミノ酸も特異的に測定できる。また、形成されたアミノアシルＡＭＰ-ＮＲＰＳ複合体からＮＲＰＳ及びアミノ酸を遊離させ、それらをアミノアシルＡＭＰ-ＮＲＰＳ複合体の形成に繰り返し利用することによって、測定対象であるピロリン酸等の反応産生物を、試料中に含まれるＮＲＰＳ及び／又は、アミノ酸より多くのモル数まで産生させることができる。その結果、従来技術に比べてより簡便な手段でこれら反応産生物を測定する場合であっても、従来技術の多段階酵素反応を用いた蛍光法などによる高感度分析のアミノ酸定量法のアミノ酸定量範囲と同等の範囲での定量が可能となる。更に、同じ測定手段を用いる場合には、より低濃度範囲のアミノ酸の定量が可能である。 In the amino acid quantification method according to the present invention, proteinaceous amino acids (20 kinds of L-type amino acids constituting a protein) can be specifically measured, and D-type amino acids which are isomers, and further methylation, Modified amino acids such as acylation and glycosylation and non-proteinaceous amino acids such as ornithine can also be specifically measured. Further, by releasing NRPS and amino acids from the formed aminoacyl AMP-NRPS complex and repeatedly using them for the formation of the aminoacyl AMP-NRPS complex, a reaction product such as pyrophosphate to be measured can be used as a sample. It is possible to produce up to a larger number of moles than the NRPS and / or amino acids contained therein. As a result, even when these reaction products are measured by a simpler means than the conventional technique, the amino acid quantification by the amino acid quantification method of high-sensitivity analysis by the fluorescence method using the multi-step enzyme reaction of the prior art. It is possible to quantify in the same range as the range. Furthermore, when the same measuring means is used, amino acids in a lower concentration range can be quantified.

更に、本発明によって、ＮＲＰＳを用いた、測定対象のタンパク質性アミノ酸、また、Ｄ型アミノ酸、修飾アミノ酸及びオルニチン等の非タンパク質性アミノ酸を特異的且つ簡便、高感度に定量する方法及びアミノ酸定量用キットを提供できる。 Furthermore, according to the present invention, a method for specifically, easily, and highly sensitively quantifying proteinaceous amino acids to be measured and nonproteinogenic amino acids such as D-type amino acids, modified amino acids, and ornithine using NRPS, and for amino acid quantification. Kits can be provided.

ＮＲＰＳ反応を示す図である。It is a figure which shows the NRPS reaction. 本発明の反応工程を示す図である。It is a figure which shows the reaction process of this invention. 各種ＮＲＰＳ反応により生じるピロリン酸産生量を示す図である。It is a figure which shows the amount of pyrophosphate produced by various NRPS reactions. 各種ＡＴＰ及び酵素濃度によるピロリン酸産生量を示す図である。It is a figure which shows the amount of pyrophosphate production by various ATP and enzyme concentration. 二価陽イオンの種類によるピロリン酸産生量を示す図である。It is a figure which shows the amount of pyrophosphate production by the type of a divalent cation. 各種二価陽イオン濃度によるピロリン酸産生量を示す図である。It is a figure which shows the amount of pyrophosphate produced by the concentration of various divalent cations. 各ｐＨにおけるピロリン酸産生量を示す図である。It is a figure which shows the amount of pyrophosphate production at each pH. 各温度におけるピロリン酸産生量を示す図である。It is a figure which shows the amount of pyrophosphate production at each temperature. 本発明と公知文献（非特許文献７）のＮＲＰＳ反応におけるピロリン酸産生量を示す図である。It is a figure which shows the amount of pyrophosphate production in the NRPS reaction of this invention and a publicly known document (Non-Patent Document 7). モリブデンブルー法によるピロリン酸測定におけるアミノ酸検量線を示す図である。It is a figure which shows the amino acid calibration curve in the measurement of pyrophosphate by the molybdenum blue method.

本発明方法の反応１及び反応３では、アミノ酸、該アミノ酸に対応するＮＲＰＳ、及びＡＴＰを反応させて、アミノアシルＡＭＰ-ＮＲＰＳ複合体を形成させる。本発明方法に使用するＮＲＰＳは、各アミノ酸に対し特異的に作用する当業者に公知の任意のＮＲＰＳを用いることができる。例えば、オルニチン（Ｏｒｎ）であれば、オルニチンに特異的に作用するＮＲＰＳ、リジン（Ｌｙｓ）であれば、リジンに特異的に作用するＮＲＰＳなどが挙げられる。また、本発明に使用するＮＲＰＳは、例えば、Ｓｔｒｅｐｔｏｍｙｃｅｓ属、Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ属、Ｐｓｅｕｄｏｍｏｎａｓ属、Ａｓｐｅｒｇｉｌｌｕｓ属、Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ属、及びＬｅｃａｎｉｃｉｌｌｕｍ属などの微生物由来など、各種生物由来のＮＲＰＳであれば、いずれのＮＲＰＳでも良く、特に取扱及び生産性の面の観点から、微生物由来のＮＲＰＳが好ましい。ＮＲＰＳは、Ａドメイン、Ｔドメイン及びＣドメインから構成されるが、好ましくは、Ａドメインのみから構成されるもの、並びに/又は、ＡドメインとＴドメインから構成されるものが良い。また、組換え型ＮＲＰＳでも良く、合成したＮＲＰＳでも良い。可溶性酵素が好ましいが、不溶性酵素に界面活性剤を組み合わせても良く、可溶化タンパクとの融合又は膜結合部分の削除等により不溶性酵素を可溶化させた酵素でも良い。ＮＲＰＳの公知のアミノ酸配列を利用でき、Ａドメインから構成される組換え型のＮＲＰＳは、６０％、７０％、８０％、９０％又は９５％以上の同一性を有する配列を有し、また、ＡドメインとＴドメインから構成される組換え型のＮＲＰＳは、６０％、７０％、８０％、９０％又は９５％以上の同一性を有する配列を有し、ＮＲＰＳ活性を有する蛋白質を使用しても良い。 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 complex. As the NRPS used in the method of the present invention, any NRPS known to those skilled in the art that acts specifically on each amino acid can be used. For example, in the case of ornithine (Orn), NRPS that acts specifically on ornithine, and in the case of lysine (Lys), NRPS that acts specifically on lysine can be mentioned. Further, the NRPS used in the present invention may be any NRPS derived from various organisms such as those derived from microorganisms such as Streptomyces, Corynebacterium, Pseudomonas, Aspergillus, Saccharoporyspora, and Lecanicillum. NRPS derived from microorganisms is preferable, particularly from the viewpoint of handling and productivity. The NRPS is composed of A domain, T domain and C domain, but preferably one composed of only A domain and / or one composed of A domain and T domain. Further, it may be a recombinant NRPS or a synthesized NRPS. A soluble enzyme is preferable, but an insoluble enzyme may be combined with a surfactant, or an enzyme in which the insoluble enzyme is solubilized by fusion with a solubilized protein or removal of a membrane-bound portion may be used. A known amino acid sequence of the NRPS can be utilized, and the recombinant NRPS composed of the A domain has a sequence having 60%, 70%, 80%, 90% or 95% or more identity, and also Recombinant NRPS composed of A domain and T domain has a sequence having 60%, 70%, 80%, 90% or 95% or more identity, and uses a protein having NRPS activity. Is also good.

本発明に使用するＮＲＰＳの調製方法としては、当業者に公知の任意の方法・手段、例えば、ＮＲＰＳを含む対象物に加水し、粉砕機、超音波破砕機などで粉砕後、破砕した破砕物を遠心分離、濾過などで固形物を取り除いた抽出物、さらに当該抽出物をカラムクロマトグラフィーなどにより精製、単離したＮＲＰＳなどを用いることができる。即ち、本発明の主な技術的特徴は、ＮＲＰＳを用いるアミノ酸定量方法に於いて、形成されたアミノアシルＡＭＰ-ＮＲＰＳ複合体中からＮＲＰＳ及びアミノ酸を遊離させて、それらをアミノアシルＡＭＰ-ＮＲＰＳ複合体の形成に繰り返し利用することによって、測定対象であるピロリン酸等の反応産生物を、試料中に含まれるＮＲＰＳ及び／又はアミノ酸より多くのモル数まで産生させることであり、ＮＲＰＳの調製方法は何ら限定されるものではない。 As a method for preparing NRPS used in the present invention, any method / means known to those skilled in the art, for example, a crushed product obtained by adding water to an object containing NRPS, crushing it with a crusher, an ultrasonic crusher, or the like, and then crushing it. An extract from which solid matter has been removed by centrifugation, filtration or the like, and NRPS obtained by 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 the amino acid quantification method using NRPS, NRPS and amino acids are released from the formed aminoacyl AMP-NRPS complex, and they are released into the aminoacyl AMP-NRPS complex. By repeatedly using it for formation, a reaction product such as pyrophosphate to be measured is produced up to a number of moles larger than the NRPS and / or amino acid contained in the sample, and the method for preparing the NRPS is limited. It is not something that is done.

本発明の測定対象となるアミノ酸は、Ｌ-リジン、Ｌ-ロイシン及びＬ-イソロイシンなどのＬ型の２０種類のタンパク質性アミノ酸、Ｌ型アミノ酸の異性体であるＤ-リジン、Ｄ-ロイシン及びＤ-イソロイシンなどのＤ型のアミノ酸、オルニチン、β-リジン及びα-メチル-Ｌセリンなど非タンパク質性アミノ酸でも良い。 The amino acids to be measured in the present invention are 20 kinds of L-type proteinaceous amino acids such as L-lysine, L-leucine and L-isoleucine, and D-lysine, D-leucine and D which are isomers of L-type amino acids. -D-type amino acids such as isoleucine and non-protein amino acids such as ornithine, β-lysine and α-methyl-L serine may be used.

また、本発明に使用する試料に特に制限はなく、例えば、血液、生鮮食品、加工食品及び飲料などがあげられる。各試料中のアミノ酸濃度は、各アミノ酸により異なる。例えば、血液中のアミノ酸濃度は、イソロイシンは、４１〜８５ｎｍｏｌ／ｍＬ、ロイシンは、８１〜１５４ｎｍｏｌ／ｍＬ、バリンは、１５８〜２８８ｎｍｏｌ／ｍＬ、リジンは、１１９〜２５７ｎｍｏｌ／ｍＬ、オルニチンは、４３〜９６ｎｍｏｌ／ｍＬなどである。生鮮食品中の遊離アミノ酸含量は、例えば、ニンニクでは、リジンは、５ｍｇ／１００ｇ、アルギニンは、１３６ｍｇ／１００ｇなどである。加工食品や飲料中の遊離アミノ酸含量は、例えば、醤油では、リジンは、２１３ｍｇ／１００ｇ、アラニンは、３４８ｍｇ／１００ｇ、ロイシンは、４５０ｍｇ／１００ｇなどである。コーヒー生豆ロブスタ種の完熟では、アスパラギン酸は、７６ｍｇ／１００ｇ、ロイシンは、６ｍｇ／１００ｇ、バリンは、１０ｍｇ／１００ｇなどである。これら各試料中の予想されるアミノ酸濃度によって、適宜、希釈調製し、本発明の試料として使用できる。 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 to 85 nmol / mL for isoleucine, 81 to 154 nmol / mL for leucine, 158 to 288 nmol / mL for valine, 119 to 257 nmol / mL for lysine, and 43 to 257 nmol / mL for lysine. For example, 96 nmol / mL. The free amino acid content in fresh foods is, for example, 5 mg / 100 g of lysine and 136 mg / 100 g of arginine in garlic. For example, in soy sauce, the content of free amino acids in processed foods and beverages is 213 mg / 100 g for lysine, 348 mg / 100 g for alanine, 450 mg / 100 g for leucine, and the like. In the ripeness of Robusta coffee beans, aspartic acid is 76 mg / 100 g, leucine is 6 mg / 100 g, valine is 10 mg / 100 g, and the like. Depending on the expected amino acid concentration in each of these samples, it can be appropriately diluted and prepared and used as the sample of the present invention.

本発明に使用する試料中のアミノ酸には、Ｌ体及びＤ体が混在してもよい。そのような場合には、例えば、本発明方法における前処理として、Ｌ体又はＤ体のアミノ酸の何れか一方を除去するのが好ましい。Ｌ体又はＤ体の何れか一方を除去する方法として、カラムクロマトグラフィー又は適当な酵素によるＬ体又はＤ体のアミノ酸の何れか一方を除去する等、当業者に公知の任意の方法が挙げられる。尚、生体試料には、Ｌ体及びＤ体が混在するが、哺乳類体内中の殆どのＤ体アミノ酸は、Ｌ体アミノ酸の０.１〜１.０％程度、果物では、Ｄ体アミノ酸は、Ｌ体アミノ酸の３.０％以下であり、生体試料中のＤ体アミノ酸含量は、Ｌ体アミノ酸の定量に影響を与えるほど含有していない。 The amino acids in the sample used in the present invention may be a mixture of L-form and D-form. In such a case, for example, as a pretreatment in the method of the present invention, it is preferable to remove either the L-form or D-form amino acid. Examples of the method for removing either the L-form or the D-form include any method known to those skilled in the art, such as column chromatography or removal of either the L-form or D-form amino acid by an appropriate enzyme. .. In addition, although L-form and D-form are mixed in the biological sample, most D-form amino acids in the mammalian body are about 0.1 to 1.0% of the L-form amino acid, and in fruits, the D-form amino acid is. It is 3.0% or less of the L-amino acid, and the D-amino acid content in the biological sample is not enough to affect the quantification of the L-amino acid.

当該反応に使用される反応液中のＮＲＰＳ濃度は、試料の種類、推定される試料中のアミノ酸濃度、ＡＴＰ濃度、及び、反応時間・温度等の各種反応条件に応じて、当業者が適宜決められる。ＮＲＰＳ濃度を高濃度することによって反応を短時間に完了させることが出来、逆に反応時間が長時間でも良い場合は、ＮＲＰＳ濃度は低濃度で良い。例えば、Ｓｔｒｅｐｔｏｍｙｃｅｓ属、Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ属及びＣｏｒｙｎｅｂａｃｔｅｒｉｕｍ属などの放線菌由来のＮＲＰＳの濃度は、０．１μＭ以上、より好ましくは０．５μＭ以上、さらに好ましくは１．０μＭ以上、特に好ましくは５．０μＭ以上、最も好ましくは１０.０μＭ以上とすることができる。いずれにしても、本発明方法では、ＮＲＰＳを繰り返し使用されるので、予想される試料中のアミノ酸量に対して、過剰量のＮＲＰＳを添加する必要はない、という利点を有する。従って、ＮＲＰＳ濃度の上限は、経済性なども考慮して当業者が適宜設定することが出来る。 The NRPS concentration in the reaction solution used for the reaction is appropriately determined by those skilled in the art according to various reaction conditions such as the type of sample, the estimated amino acid concentration in the sample, the ATP concentration, and the reaction time and temperature. Be done. When the reaction can be completed in a short time by increasing the NRPS concentration to a high concentration, and conversely, when the reaction time may be long, the NRPS concentration may be low. For example, the concentration of NRPS derived from actinomycetes such as Streptomyces, Saccharoporyspora and Corynebacterium is 0.1 μM or more, more preferably 0.5 μM or more, still more preferably 1.0 μM or more, and particularly preferably 5.0 μM or more. Most preferably, it can be 10.0 μM or more. In any case, since the NRPS is used repeatedly in the method of the present invention, there is an advantage that it is not necessary to add an excessive amount of NRPS to the expected amount of amino acids in the sample. Therefore, the upper limit of the NRPS concentration can be appropriately set by those skilled in the art in consideration of economic efficiency and the like.

本発明方法の反応１又は反応３ではＮＲＰＳと共にＡＴＰ、二価陽イオンを用いる。本発明に使用するＡＴＰは、ナトリウム塩、リチウム塩などが使用できる。当該反応に使用される反応液中のＡＴＰの濃度は、試料の種類、予想される試料中のアミノ酸濃度、ＮＲＰＳ濃度、ヌクレオチド濃度、及び、反応時間・温度等の各種反応条件に応じて、当業者が適宜決められるが、予想される試料中のアミノ酸濃度に対して過剰となるように添加するのが好ましい。例えば試料が血液の場合、ＡＴＰ濃度は、０.０１ｍＭ以上、より好ましくは０.１ｍＭ以上、さらに好ましくは１.０ｍＭ以上、特に好ましくは１０.０ｍＭ以上、最も好ましくは４０.０ｍＭ以上とすることができる。ＡＴＰ濃度の上限は、経済性なども考慮して当業者が適宜設定することが出来る。また、本発明に使用する二価陽イオンは、マグネシウム、マンガン、コバルト、カルシウムなどが使用できる。二価陽イオンは、ＮＲＰＳにより要求性が異なるため、使用するＮＲＰＳに適した二価陽イオンを適宜使用すれば良い。当該反応に使用される反応液中の二価陽イオンの濃度は、適宜決められるが、ＡＴＰ濃度に対して同等以上に添加するのが好ましい。例えば、ＮＲＰＳにおけるＡＴＰ：二価陽イオンの比率は、少なくとも１：１０、より好ましくは少なくとも１：７、さらに好ましくは少なくとも１：５、特に好ましくは少なくとも１：３、最も好ましくは少なくとも１：１とすることができる。 In reaction 1 or reaction 3 of the method of the present invention, ATP and divalent cation are used together with NRPS. As the ATP used in the present invention, sodium salt, lithium salt and the like can be used. The concentration of ATP in the reaction solution used for the reaction depends on the type of sample, the expected amino acid concentration in the sample, the NRPS concentration, the nucleotide concentration, and various reaction conditions such as reaction time and temperature. The trader will be determined as appropriate, but it is preferable to add the amino acid in excess of the expected amino acid concentration in the sample. For example, when the sample is blood, the ATP concentration should be 0.01 mM or more, more preferably 0.1 mM or more, still more preferably 1.0 mM or more, particularly preferably 10.0 mM or more, and most preferably 40.0 mM or more. 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. Further, as the divalent cation used in the present invention, magnesium, manganese, cobalt, calcium and the like can be used. Since the divalent cation has different requirements depending on the NRPS, a divalent cation suitable for the NRPS to be used may be appropriately used. The concentration of divalent cations in the reaction solution used for the reaction is appropriately determined, but it is preferable to add the divalent cations in an amount equal to or higher than the ATP concentration. For example, the ratio of ATP: divalent cations in the NRPS is at least 1:10, more preferably at least 1: 7, still more preferably at least 1: 5, particularly preferably at least 1: 3, and most preferably at least 1: 1. Can be.

続いて、本発明方法の反応２では、反応１及び／又は反応３で形成させたアミノアシルＡＭＰ-ＮＲＰＳ複合体に対してアミノ酸再生試薬が作用し、該複合体からＮＲＰＳ及びアミノ酸が遊離する。当該反応のアミノ酸再生試薬としては、ＡＴＰ、アデノシン二リン酸（ＡＤＰ）、アデノシン一リン酸（ＡＭＰ）、グアノシン三リン酸（ＧＴＰ）、デオキシアデノシン三リン酸（ｄＡＴＰ）などから任意に選択される一種又はそれらの組み合わせからなるヌクレオチドや、水酸化ナトリウム、水酸化カリウム、炭酸ナトリウム及び緩衝剤などの水酸化物イオンを発生するアルカリ性化合物を使用できる。反応２に於いては、アミノアシルＡＭＰ-ＮＲＰＳ複合体からのＮＲＰＳ及びアミノ酸の遊離と共に、使用するアミノ酸再生試薬に応じて、ＡＭＰ、Ａｐ４Ａ等が生成される。当該反応に使用される反応液中のアミノ酸再生試薬の濃度は、試料の種類、予想される試料中のアミノ酸濃度、ＮＲＰＳ濃度、ＡＴＰ濃度及び反応時間・温度等の各種反応条件に応じて、当業者が適宜決められるが、ＡＭＰ、Ａｐ４Ａ等の生成に於いて消費されるので、ヌクレオチドは試料中のアミノ酸濃度に対して過剰となるように添加するのが好ましい。例えば、試料が血液の場合、ヌクレオチド濃度は、０.０１ｍＭ以上、より好ましくは０.１ｍＭ以上、さらに好ましくは１.０ｍＭ以上、特に好ましくは１０.０ｍＭ以上、最も好ましくは４０.０ｍＭ以上とすることができる。また、アルカリ性化合物は、反応の場（反応系）をｐＨ７以上にできれば良い。例えば、Ｓｔｒｅｐｔｏｍｙｃｅｓ属、Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ属及びＣｏｒｙｎｅｂａｃｔｅｒｉｕｍ属などの放線菌由来のＮＲＰＳでは、好ましくはｐＨ７.０以上、より好ましくはｐＨ７.５以上、さらに好ましくは、ｐＨ８.０以上とする。尚、反応ｐＨは、当業者に公知の任意の当業者に公知の任意の緩衝剤である、ＨＥＰＥＳバッファー、ＣＨＥＳバッファー、ＴＲＩＳバッファー、ＭＯＰＳバッファーなどを使用して調整することが出来る。従って、本発明方法では、ＮＲＰＳを再び反応可能な状態とさせるために、特許文献３に記載されているようなアミン類等のアミノアシルＡＭＰ-ＮＲＰＳ複合体分解試薬を添加する必要がなく、更に、遊離したアミノ酸が該試薬と反応することがないので、遊離したアミノ酸をアミノアシルＡＭＰ-ＮＲＰＳ複合体の形成に再び利用することができる。 Subsequently, in the reaction 2 of the method of the present invention, the amino acid regeneration reagent acts on the aminoacyl AMP-NRPS complex formed in the reaction 1 and / or the reaction 3, and the NRPS and the amino acid are released 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 or a combination thereof, and alkaline compounds that generate hydroxide ions such as sodium hydroxide, potassium hydroxide, sodium carbonate, and buffers can be used. In Reaction 2, along with the release of NRPS and amino acids from the aminoacyl AMP-NRPS complex, AMP, Ap4A and the like are produced depending on the amino acid regeneration reagent used. The concentration of the amino acid regeneration reagent in the reaction solution used for the reaction depends on various reaction conditions such as the type of sample, the expected amino acid concentration in the sample, the NRPS concentration, the ATP concentration, and the reaction time / temperature. Although the trader is appropriately determined, it is preferable to add the nucleotide in excess of the amino acid concentration in the sample because it is 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 more, more preferably 0.1 mM or more, still more preferably 1.0 mM or more, particularly preferably 10.0 mM or more, and most preferably 40.0 mM or more. be able to. Further, the alkaline compound may have a reaction field (reaction system) of pH 7 or higher. For example, the NRPS derived from actinomycetes such as Streptomyces, Saccharoporyspora and Corynebacterium is preferably pH 7.0 or higher, more preferably pH 7.5 or higher, and even more preferably pH 8.0 or higher. The reaction pH can be adjusted by using any buffer known to those skilled in the art, such as HEPES buffer, CHES buffer, TRIS buffer, and MOPS buffer. Therefore, in the method of the present invention, it is not necessary to add an aminoacyl AMP-NRPS complex decomposition reagent such as amines as described in Patent Document 3 in order to make the NRPS reactive again, and further. Since the free amino acid does not react with the reagent, the free amino acid can be reused for the formation of the aminoacyl AMP-NRPS complex.

本発明方法の反応３では、反応２で遊離したアミノ酸及び/又はＮＲＰＳを反応１において再び使用することによってアミノアシルＡＭＰ-ＮＲＰＳ複合体反応を形成させる。更に、本発明方法の工程（Ｉ）において、当業者に公知の任意の方法によって、反応１又は３で生じたピロリン酸及び反応系中のＡＭＰにホスホエノールピルビン酸とピルビン酸ジキナーゼとを反応させることによって産生されるＡＴＰを、アミノアシルＡＭＰ-ＮＲＰＳ複合体形成及び該複合体からのＮＲＰＳ及びアミノ酸の遊離に再利用すること、及び/又は、反応２でヌクレオチドから生成されるＡｐ４Ａに対しＡｐ４Ａピロホスホヒドラーゼを作用させることによって産生されるＡＤＰを、反応２におけるヌクレオチドとして再利用することもできる。 In reaction 3 of the method of the present invention, the amino acid and / or NRPS released in reaction 2 is used again in reaction 1 to form an aminoacyl AMP-NRPS complex reaction. Further, in step (I) of the method of the present invention, phosphoenolpyruvate and pyruvate diphosphate are reacted with pyrophosphate generated in reaction 1 or 3 and AMP in the reaction system by any method known to those skilled in the art. The ATP produced by this is reused for aminoacyl AMP-NRPS complex formation and release of NRPS and amino acids from the complex, and / or Ap4A pyrophospho for Ap4A produced from nucleotides in Reaction 2. The ADP produced by the action of hydrase can also be reused as a nucleotide in Reaction 2.

例えば、反応２で作用するヌクレオチドとしてＡＴＰを選択し、更に、上記のような反応を利用してＡＴＰの再産生をしない場合には、本明細書の実施例に示されるように、工程（Ｉ）の反応系全体で、上記に示したＡＴＰ及びヌクレオチドの夫々の濃度を合計した、より高濃度のＡＴＰを添加することが好ましい。 For example, when ATP is selected as the nucleotide acting in reaction 2 and ATP is not reproduced by utilizing the reaction as described above, the step (I) is as shown in the examples of the present specification. ), It is preferable to add a higher concentration of ATP, which is the sum of the concentrations of ATP and nucleotides shown above.

以上の結果、前述のアミノ酸再生試薬（ＡＴＰなどのヌクレオチド及び/又はアルカリ性化合物）及び該アミノ酸に対応するＮＲＰＳの組成等の反応条件において、工程（Ｉ）に於ける反応の結果、ピロリン酸及び/又は水素イオン等の夫々の反応産生物の各々について、試料中に含まれているＮＲＰＳ及び／又は、測定対象のアミノ酸のモル数より多いモル数の分子が産生され得る。その結果、本発明方法では、従来技術より極めて低いアミノ酸濃度から定量可能となる。従って、この点は従来技術と比較して本発明の顕著な効果といえる。 As a result of the above, under the reaction conditions such as the composition of the above-mentioned amino acid regeneration reagent (nucleotide and / or alkaline compound such as ATP) and NRPS corresponding to the amino acid, as a result of the reaction in step (I), pyrophosphate and / Alternatively, for each of the reaction products such as hydrogen ions, molecules having a number of moles larger than the number of moles of the NRPS and / or the amino acid to be measured contained in the sample can be produced. As a result, in the method of the present invention, it is possible to quantify from an amino acid concentration extremely lower than that 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.

しかしながら、当該反応条件下で産生されるピロリン酸等の反応産生物が、試料中のアミノ酸のモル数以下の量であっても、該反応産生物に基づきアミノ酸の定量が可能であれば、ピロリン酸等の反応産生物が当該アミノ酸のモル数以上に産生される必要はない。従って、本発明の工程（Ｉ）に於いて産生されるピロリン酸や水素イオンの量は、工程（ＩＩ）に於ける該反応産生物の適当な測定方法によってアミノ酸の定量が可能となる限り、特に限定されない。また、本発明方法の（工程Ｉ−４）に於ける、反応２（工程Ｉ−２）及び反応３（工程Ｉ−３）の繰り返しの回数についても、工程（ＩＩ）に於ける該反応産生物の適当な測定方法によってアミノ酸の定量が可能となる限り、特に制限はない。 However, even if the amount of the reaction product such as pyrophosphate produced under the reaction conditions is less than the number of moles of the amino acid in the sample, if the amino acid can be quantified based on the reaction product, the pyrolin Reaction products such as acids need not be produced in excess of the number of moles of the amino acid. Therefore, the amount of pyrophosphate and hydrogen ions produced in the step (I) of the present invention is as long as the amino acids can be quantified by an appropriate measurement method of the reaction product in the step (II). There is no particular limitation. Further, the number of repetitions of the reaction 2 (step I-2) and the reaction 3 (step I-3) in the method of the present invention (step I-4) is also the reaction product in the step (II). There are no particular restrictions as long as amino acids can be quantified by an appropriate measurement method for living organisms.

本発明方法の工程（Ｉ）における反応温度は、各反応が生じるような任意の温度で良い。例えば、Ｓｔｒｅｐｔｏｍｙｃｅｓ属，Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ属及びＣｏｒｙｎｅｂａｃｔｅｒｉｕｍ属等の放線菌由来のＮＲＰＳの場合、好ましくは４〜５０℃、より好ましくは１０〜４０℃、最も好ましくは２０〜３５℃とすることが適している。また、当該反応時間も試料中のアミノ酸とＮＲＰＳ反応が生じるような任意の時間で良いが、好ましくは５〜１２０分程度、より好ましくは１０〜９０分程度、さらに好ましくは、１５〜６０分程度であることが望ましい。 The reaction temperature in step (I) of the method 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, Saccharoporyspora and Corynebacterium, the temperature is preferably 4 to 50 ° C, more preferably 10 to 40 ° C, and most preferably 20 to 35 ° C. The reaction time may be any time such that an NRPS reaction occurs with the amino acid in the sample, but is preferably about 5 to 120 minutes, more preferably about 10 to 90 minutes, and further preferably about 15 to 60 minutes. Is desirable.

本発明方法の工程（Ｉ）に含まれる各工程で使用する試薬・酵素等の各反応成分は、ＮＲＰＳ反応が生じる添加方法である限り、当業者に公知の任意の手段・手順等で反応系に添加することができる。例えば、各成分を反応開始前に一度に反応系に予め添加するか、又は、ＮＲＰＳ又はアミノ酸を最後に添加し反応させても良い。 Each reaction component such as a reagent or an enzyme used in each step included in the step (I) of the method of the present invention is a reaction system by any means / procedure known to those skilled in the art as long as it is an addition method that causes an NRPS reaction. Can be added to. For example, each component may be added to the reaction system at once before the start of the reaction, or NRPS or an amino acid may be added last to cause the reaction.

本発明方法の工程（ＩＩ）では、工程（Ｉ）で生じたピロリン酸及び水素イオン等の各反応産生物の夫々の量を測定し、該反応産生物の測定量に基づきアミノ酸の量を決定する。工程（ＩＩ）は、測定方法等に応じて、工程（Ｉ）に於ける試料中のアミノ酸とＮＲＰＳとの反応を、例えば、実施例に記載されているように、トリクロロ酢酸を反応系に添加する等の当業者に公知の任意の方法・手段で停止させた後、あるいは、工程（Ｉ）に於ける反応が進行中の任意の段階で適宜、実施することが出来る。 In step (II) of the method of the present invention, the amount of each reaction product such as pyrophosphate and hydrogen ion generated in step (I) is measured, and the amount of amino acid is determined based on the measured amount of the reaction product. To do. In step (II), depending on the measurement method and the like, the reaction between the amino acid in the sample and NRPS in step (I) is added to the reaction system, for example, as described in Examples. After stopping by any method / means known to those skilled in the art, or at any stage during which the reaction in step (I) is in progress, it can be appropriately carried out.

本発明の工程（Ｉ）で生じたピロリン酸量の測定には、当業者に公知の任意の方法・手段、例えば、モリブデン酸とピロリン酸を反応させ産生した青色の錯体の吸光度を測定するモリブデンブルー法、ヒポキサンチン−グアニンホスホリボシルトランスフェラーゼ、キサンチンオキシダーゼ又はキサンチンデヒドロゲナーゼを組みわせる方法、ルミノールと無機ピロホスファターゼ、ピルビン酸オキシダーゼ及びペルオキシダーゼを組み合わせ産生物の発光を測定する方法などのピロリン酸を測定できる酵素法などが使用できる。また、酵素反応などで酸化還元反応を起こし、その酸化還元反応に由来する電流値を検出する多電極電位計測計により電位変化を測定する測定方法などを使用することができる。さらに、当該反応で産生された水素イオンの測定には、水素イオンを検出するガラス電極やイオン感応性電界効果トランジスタにより電位変化を測定する測定方法などを使用することができる。本発明の工程（Ｉ）で生じたピロリン酸、水素イオンなどは、反応溶液から分離し、測定することができる。反応溶液からのピロリン酸、水素イオンなどの分離方法としては、測定に影響の無い方法であれば特に限定されないが、例えば、酸処理による除タンパク質、ペーパークロマトグラフィー分離、マイクロ流体デバイスでの分離などが挙げられる。本発明の主な技術的特徴は、ＮＲＰＳを用いるアミノ酸定量方法に於いて、形成されたアミノアシルＡＭＰ-ＮＲＰＳ複合体中からＮＲＰＳ及びアミノ酸を遊離させて、これら化合物を、再度、該複合体の形成に繰り返し利用することにより、測定対象であるピロリン酸等の反応産生物を、試料中に含まれるＮＲＰＳ及び／又は、アミノ酸より多くのモル数まで産生させることであり、反応産生物の量の測定方法は何ら限定されるものではない。 For the measurement of the amount of pyrophosphoric acid produced in the step (I) of the present invention, any method / means known to those skilled in the art, for example, molybdenum for measuring the absorbance of the blue complex produced by reacting molybdic acid with pyrophosphoric acid. Pyrophosphoric acid can be measured by the blue method, the method of combining hypoxanthine-guanine phosphoribosyl transferase, xanthine oxidase or xanthine dehydrogenase, the method of combining luminol with inorganic pyrophosphatase, pyruvate oxidase and peroxidase, and measuring the luminescence of the product. Enzyme method etc. can be used. Further, a measuring method or the like in which a redox reaction is caused by an enzymatic reaction or the like and the potential change is measured by a multi-electrode potential measuring meter that detects the current value derived from the redox reaction can be used. Further, for the measurement of the hydrogen ions produced in the reaction, a measuring method of measuring the potential change by a glass electrode for detecting the hydrogen ions or an ion-sensitive electric field effect transistor can be used. Pyrophosphoric acid, hydrogen ions, etc. generated in the 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 does not affect the measurement, but for example, protein removal by acid treatment, paper chromatography separation, separation with a microfluidic device, etc. Can be mentioned. The main technical feature of the present invention is that in an amino acid quantification method using NRPS, NRPS and amino acids are released from the formed aminoacyl AMP-NRPS complex, and these compounds are recombined to form the complex. By repeatedly using the reaction product, a reaction product such as pyrophosphate to be measured is produced up to a number of moles larger than the NRPS and / or amino acid contained in the sample, and the amount of the reaction product is measured. The method is not limited in any way.

本発明は、上記の本発明方法を実施するための、試料中のアミノ酸定量に必要な前述の各成分を含む、アミノ酸定量用キットを提供する。当該キットは、安定化剤又は緩衝剤等の当業者に公知の他の任意成分を適宜含有させ、前記酵素等試薬成分の安定性を高めても良い。測定に影響の無い成分であれば特に限定されないが、例えば、牛血清アルブミン（ＢＳＡ）、卵白アルブミン、糖類、糖アルコール類、カルボキシル基含有化合物、酸化防止剤、界面活性剤、又は酵素と作用性のないアミノ酸類等を例示できる。また、当該キットの一例として前述のピロリン酸や水素イオンを測定するためのキットを挙げることが出来る。 The present invention provides an amino acid quantification kit containing the above-mentioned components necessary for quantifying amino acids in a sample for carrying out the above-mentioned method of the present invention. 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 the reagent components such as enzymes. It is not particularly limited as long as it is a component that does not affect the measurement, but is effective with, for example, bovine serum albumin (BSA), egg white albumin, saccharides, sugar alcohols, carboxyl group-containing compounds, antioxidants, surfactants, or enzymes. Examples of amino acids that do not have Further, as an example of the kit, the above-mentioned kit for measuring pyrophosphoric acid and hydrogen ions can be mentioned.

以下、実施例によって本発明を具体的に説明するが、本発明の技術的範囲は以下の実施例によって限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to Examples, but the technical scope of the present invention is not limited to the following Examples.

（酵素の調製）
放線菌（Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ属又はＣｏｒｙｎｅｂａｃｔｅｒｉｕｍ属）由来のＮＲＰＳ配列（Ｐｌｓ-ｓｅ-Ａドメイン、Ｐｌｓ-ｃｇ-Ａドメイン又はＰｌｓ-ｃｇ-ＡＴドメイン）をもつプラスミドで大腸菌ＪＭ１０９を形質転換し、発現株として用いた。各発現株についてアンピシリンを終濃度２００μｇ/ｍＬを含むＬＢ培地により３７℃で培養後、ＯＤ６６０＝０．６-０．８に達したところで、培養液を氷水で冷却し、終濃度０．１ｍＭとなるようにＩＰＴＧを添加した。培養液を１８℃で終夜培養後、集菌を行い、得られた菌体を超音波破砕し、無細胞抽出液を調製した。さらに遠心分離を行い、得られた上清の一部を用いて電気泳動法により目的酵素の発現を確認した。次いで残りの上清をＨｉｓタグカラム（商品名：ＴＡＬＯＮ ｓｕｐｅｒｆｌｏｗ、ＧＥヘルスケア製）により夾雑タンパクを除去することにより、Ｌ-オルニチンに特異的に作用するＡドメインから成るＮＲＰＳのＰｌｓ-ｓｅ-Ａ、Ｌ-リジン及びＤ-リジンに特異的に作用するＡドメインから成るＮＲＰＳのＰｌｓ-ｃｇ-Ａ、Ｌ-リジン及びＤ-リジンに特異的に作用するＡドメイン及びＴドメイン（以下、ＡＴドメイン）から成るＮＲＰＳのＰｌｓ-ｃｇ-ＡＴを得た。(Preparation of enzyme)
Escherichia coli JM109 is 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 Saccharoporyspora or Corynebacterium) and used as an expression strain. There was. For each expression strain, ampicillin was cultured in LB medium containing a final concentration of 200 μg / mL at 37 ° C., and when OD660 = 0.6-0.8 was reached, the culture solution was cooled with ice water to a final concentration of 0.1 mM. IPTG was added so as to become. After culturing the culture solution at 18 ° C. overnight, the cells were collected and the obtained cells were ultrasonically crushed to prepare a cell-free extract. Further centrifugation was performed, and the expression of the target enzyme was confirmed by electrophoresis using a part of the obtained supernatant. Next, by removing the contaminating protein from the remaining supernatant with a His tag column (trade name: TALON superflow, manufactured by GE Healthcare), Pls-se-A of NRPS consisting of an A domain that specifically acts on L-ornithine, From the A domain and T domain (hereinafter referred to as AT domain) that act specifically on Pls-cg-A, L-lysine and D-lysine of NRPS consisting of the A domain that acts specifically on L-lysine and D-lysine. An NRPS Pls-cg-AT was obtained.

（各種ＮＲＰＳ反応により生じるピロリン酸の産生量：本発明方法の工程（Ｉ））
２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭnＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃、６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品（本発明品）１を調製した。(Amount of pyrophosphoric acid produced by various NRPS reactions: Step (I) of the method of the present invention)
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 actinomycetes) and treat at 30 ° C. for 60 minutes. did. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the product (product of the present invention) 1.

２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭnＣｌ２、５０μＭ Ｌ-リジン又はＤ-リジン、５．０μＭ Ｐｌｓ−ｃｇ−Ａ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃、６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品（本発明品）２、３を調製した。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-lysine or D-lysine, and 5.0 μM Pls-cg-A (derived from actinomycetes) at 30 ° C. , 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the product (product of the present invention) 2 and 3.

２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｎＣｌ２、５０μＭ Ｌ-リジン又はＤ-リジン、５．０μＭ Ｐｌｓ−ｃｇ−ＡＴ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃、６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品（本発明品）４、５を調製した。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-lysine or D-lysine, and 5.0 μM Pls-cg-AT (derived from actinomycetes) at 30 ° C. , 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the products (products of the present invention) 4 and 5.

（モリブデンブルー法によるピロリン酸の測定：本発明方法の工程（ＩＩ））
調製した実施品１〜５の反応溶液１５０μＬに１Ｍメルカプトエタノール１５μＬ、発色液（２．５％モリブデン酸アンモニウム／５Ｎ硫酸）６０μＬを混合し、室温で２０分間静置した後、５８０ｎｍの吸光度を測定した。なお、基質アミノ酸の代わりに水を添加したサンプルの吸光値を、ブランクとして各サンプルの吸光値から差し引いた値から、反応溶液中のピロリン酸量を求めた。その結果、図３に示す通り、Ａドメイン及びＡＴドメインから成るＮＲＰＳ共に、ＮＲＰＳの分子数以上、さらに各基質アミノ酸が酵素反応に使用された場合に産生されるピロリン酸量の理論値より多くのピロリン酸が産生されていた。従って、本発明の方法によれば、試料中に含まれるＮＲＰＳ及びアミノ酸分子数より多くのモル数のピロリン酸が産生されることが示された。(Measurement of Pyrophosphoric Acid by Molybdenum Blue Method: Step (II) of the Method of the Present Invention)
15 μL of 1 M mercaptoethanol and 60 μL of a color-developing solution (2.5% ammonium molybdate / 5N sulfuric acid) were mixed with 150 μL of the prepared reaction solution 1 to 5 and allowed to stand at room temperature for 20 minutes, and then the absorbance at 580 nm was measured. did. The amount of pyrophosphoric acid in the reaction solution was determined from the value obtained by subtracting the absorption value of the sample to which water was added instead of the substrate amino acid from the absorption value of each sample as a blank. As a result, as shown in FIG. 3, both the NRPS consisting of the A domain and the AT domain are equal to or more than the number of molecules of the NRPS and more than the theoretical value of the amount of pyrophosphate produced when each substrate amino acid is used in the enzymatic reaction. Pyrophosphoric acid was produced. Therefore, according to the method of the present invention, it was shown that NRPS contained in the sample and pyrophosphate having a molar number larger than the number of amino acid molecules are produced.

（各種ＡＴＰ濃度及び酵素濃度によるピロリン酸の産生量：本発明方法の工程（Ｉ））
２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、２５ｍＭ ＡＴＰ、２５ｍＭ ＭnＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭnＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−ＡＴ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃、６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品（本発明品）６、７を調製した。(Amount of pyrophosphoric acid produced by 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, 5.0 μM Pls-se-AT (derived from actinomycetes) was prepared in 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%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the products (products of the present invention) 6 and 7.

２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭnＣｌ２、５０μＭ Ｌ-リジン、１．０μＭ Ｐｌｓ−ｃｇ−Ａ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃、６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品（本発明品）８を調製した。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-lysine, 1.0 μM Pls-cg-A (derived from actinomycetes), and treat at 30 ° C. for 60 minutes. did. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the product (product of the present invention) 8.

２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、１０ｍＭ ＡＴＰ、１０ｍＭ ＭnＣｌ２、５０μＭ Ｌ-リジン、５．０μＭ Ｐｌｓ−ｃｇ−Ａ（放線菌由来）を含む反応溶液、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、２５ｍＭ ＡＴＰ、２５ｍＭ ＭnＣｌ２、５０μＭ Ｌ-リジン、５．０μＭ Ｐｌｓ−ｃｇ−Ａ（放線菌由来）を含む反応溶液、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭnＣｌ２、５０μＭ Ｌ-リジン、５．０μＭ Ｐｌｓ−ｃｇ−Ａ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃、６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品（本発明品）９、１０、１１を調製した。A 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 actinomycetes), 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 actinomycetes) 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%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the products (products of the present invention) 9, 10 and 11.

（モリブデンブルー法によるピロリン酸の測定：本発明方法の工程（ＩＩ））
実施例６、７、８で調製した実施品６〜１１のピロリン酸を実施例５に記載のモリブデンブルー法により測定した。その結果、図４に示す通り、各種ＮＲＰＳにおいてＡＴＰ又は酵素濃度の増加と共にピロリン酸の増加が認められた。また、添加したアミノ酸が全て酵素反応に使用された場合に産生されるピロリン酸量である理論値より多くのピロリン酸が産生されていた。(Measurement of Pyrophosphoric Acid by Molybdenum Blue Method: Step (II) of the Method of the Present Invention)
The pyrophosphoric acids of Examples 6 to 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 pyrophosphoric acid was observed with an increase in ATP or enzyme concentration in various NRPS. In addition, more pyrophosphoric acid was produced than the theoretical value, which is the amount of pyrophosphoric acid produced when all the added amino acids were used in the enzymatic reaction.

（二価陽イオンの種類の違いによるピロリン酸の産生量：本発明方法の工程（Ｉ））
２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｎＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｇＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＣａＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃、６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品（本発明品）１２、１３、１４を調製した。(Amount of pyrophosphoric acid produced 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 actinomycetes), 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 actinomycetes) 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%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the products (product of the present invention) 12, 13, and 14.

２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｎＣｌ２、５０μＭ Ｌ-リジン、５．０μＭ Ｐｌｓ−ｃｇ−Ａ（放線菌由来）を含む反応溶液、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｇＣｌ２、５０μＭ Ｌ-リジン、５．０μＭ Ｐｌｓ−ｃｇ−Ａ（放線菌由来）を含む反応溶液、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＣａＣｌ２、５０μＭ Ｌ-リジン、５．０μＭ Ｐｌｓ−ｃｇ−Ａ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃、６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品（本発明品）１５、１６、１７を調製した。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 actinomycetes), 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 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-lysine and 5.0 μM Pls-cg-A (derived from actinomycetes) 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%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the products (product of the present invention) 15, 16 and 17.

（モリブデンブルー法によるピロリン酸の測定：本発明方法の工程（ＩＩ））
実施例１０、１１で調製した実施品１２〜１７のピロリン酸を実施例５に記載のモリブデンブルー法により測定した。その結果、図５に示す通り、各種二価陽イオンを用いた条件で、基質アミノ酸が酵素反応に使用された場合に産生されるピロリン酸量である理論値より多くのピロリン酸が産生されていた。また、少なくとも上記実施例で使用したＮＲＰＳに関しては、Ｍｎが最適な二価陽イオンであることが示された。(Measurement of Pyrophosphoric Acid by Molybdenum Blue Method: Step (II) of the Method of the Present Invention)
The pyrophosphoric acids of Examples 12 to 17 prepared in Examples 10 and 11 were measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 5, more pyrophosphoric acid than the theoretical value, which is the amount of pyrophosphoric acid produced when the substrate amino acid is used in the enzymatic reaction, is produced under the condition using various divalent cations. It was. It was also shown that Mn is the optimal divalent cation, at least for the NRPS used in the above examples.

（二価陽イオン濃度の違いによるピロリン酸の産生量：本発明方法の工程（Ｉ））
２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｇＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、１２０ｍＭ ＭｇＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、２００ｍＭ ＭｇＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４００ｍＭ ＭｇＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃、６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品（本発明品）１８〜２１を調製した。(Amount of pyrophosphoric acid produced due to 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 actinomycetes), 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 actinomycetes) 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%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the product (product of the present invention) 18-21.

（モリブデンブルー法によるピロリン酸の測定：本発明方法の工程（ＩＩ））
実施例１３で調製した実施品１８〜２１のピロリン酸を実施例５に記載のモリブデンブルー法により測定した。その結果、図６に示す通り、二価陽イオンの濃度によって、酵素反応により産生されるピロリン酸量は異なることが示された。(Measurement of Pyrophosphoric Acid by Molybdenum Blue Method: Step (II) of the Method of the Present Invention)
The pyrophosphoric acids of Examples 18 to 21 prepared in Example 13 were measured 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 differs depending on the concentration of the divalent cation.

（反応ｐHによるピロリン酸産生量）
２００ｍＭ ＭＥＳ（ｐＨ６．０）、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ７．０）、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ７．５）、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）と、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｎＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃で６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、上清中のピロリン酸を実施例５に記載のモリブデンブルー法により測定した。その結果、図７に示す通り、ｐＨがアルカリ側になるほどピロリン酸産生量が増加し、ｐＨ８．０において、良好なピロリン酸の産生が認められた。(Amount of pyrophosphate produced 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 actinomycetes) 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%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation, and the pyrophosphoric acid in the supernatant was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 7, the amount of pyrophosphoric acid produced increased as the pH became more alkaline, and good pyrophosphoric acid production was observed at pH 8.0.

（反応ｐHによるピロリン酸産生量）
２００ｍＭ ＭＥＳ（ｐＨ６．０）、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ７．０）、又は２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）と、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｎＣｌ２、５０μＭ Ｌ-リジン、２．５μＭ Ｐｌｓ−ｃｇ−Ａ（放線菌由来）を含む反応溶液を１５０μＬ調製し、３０℃で６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、上清中のピロリン酸を実施例５に記載のモリブデンブルー法により測定した。その結果、図７に示す通り、ｐＨがアルカリ側になるほどピロリン酸産生量が増加し、ｐＨ８．０において、良好なピロリン酸の産生が認められた。(Amount of pyrophosphate produced 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 actinomycetes) 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%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation, and the pyrophosphoric acid in the supernatant was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 7, the amount of pyrophosphoric acid produced increased as the pH became more alkaline, and good pyrophosphoric acid production was observed at pH 8.0.

（反応温度によるピロリン酸産生量）
２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｎＣｌ２、５０μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液を１５０μＬ調製し、１０、又は２０、又は２５、又は３０、又は３５、又は４０℃で６０分間処理した。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、上清中のピロリン酸を実施例５に記載のモリブデンブルー法により測定した。その結果、図８に示す通り、１０〜４０℃の広い範囲において、良好なピロリン酸の産生が認められた。(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 actinomycetes), and prepare 10, or 20, or Treated at 25, 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%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation, and the pyrophosphoric acid in the supernatant was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 8, good production of pyrophosphoric acid was observed in a wide range of 10 to 40 ° C.

（本発明と公知文献記載のＮＲＰＳ反応におけるピロリン酸の産生量の比較：本発明方法の工程（Ｉ））
[比較例１]
非特許文献７記載の反応条件に従い、０．９μＭ Ｐｌｓ-ｃｇ-Ａ(放線菌由来)、５０μＭ Ｌ-リジン、５ｍＭ ＡＴＰ、５ｍＭ ＭｇＣｌ２、５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）、３２ｍＭ ヒドロキシルアミンを含む反応液３００μＬを調製し、３０℃で６０分間酵素反応を行った。酵素反応後、トリクロロ酢酸を終濃度４％となるように６０μＬ添加し反応を停止した。反応停止後、遠心分離で沈殿を除去し、比較品１を調製した。(Comparison of the amount of pyrophosphoric acid produced in the NRPS reaction described in the present invention and the publicly known document: 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. 300 μL of the containing reaction solution was prepared, and the enzymatic reaction was carried out at 30 ° C. for 60 minutes. After the enzymatic reaction, 60 μL of trichloroacetic acid was added to a final concentration of 4% to stop the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare Comparative Product 1.

[比較例２]
非特許文献７記載の反応条件に従い、０．８μＭ Ｐｌｓ-ｃｇ-ＡＴ(放線菌由来)、５０μＭ Ｌ-リジン、５ｍＭ ＡＴＰ、５ｍＭ ＭｇＣｌ２、５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）、３２ｍＭ ヒドロキシルアミンを含む反応液３００μＬを調製し、３０℃で６０分間酵素反応を行った。酵素反応後、トリクロロ酢酸を終濃度４％となるように６０μＬ添加し反応を停止した。反応停止後、遠心分離で沈殿を除去し、比較品２を調製した。[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. 300 μL of the containing reaction solution was prepared, and the enzymatic reaction was carried out at 30 ° C. for 60 minutes. After the enzymatic reaction, 60 μL of trichloroacetic acid was added to a final concentration of 4% to stop the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare Comparative Product 2.

５．０μＭ Ｐｌｓ-ｃｇ-Ａ(放線菌由来)、５０μＭ Ｌ-リジン、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｎＣｌ２、２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）を含む反応液３００μＬを調製し、３０℃で６０分間酵素反応を行った。酵素反応後、トリクロロ酢酸を終濃度４％となるように６０μＬ添加し反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品２２を調製した。Prepare 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 , and 200 mM HEPES-KOH (pH 8.0), and prepare the enzyme at 30 ° C. for 60 minutes. The reaction was carried out. After the enzymatic reaction, 60 μL of trichloroacetic acid was added to a final concentration of 4% to stop the reaction. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the product 22.

５．０μＭ Ｐｌｓ-ｃｇ-ＡＴ(放線菌由来)、５０μＭ Ｌ-リジン、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｎＣｌ２、２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）を含む反応液３００μＬを調製し、３０℃で６０分間酵素反応を行った。酵素反応後、トリクロロ酢酸を終濃度４％となるように６０μＬ添加し反応を停止した。反応停止後、遠心分離で沈殿を除去し、実施品２３を調製した。Prepare 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 , and 200 mM HEPES-KOH (pH 8.0), and enzyme at 30 ° C. for 60 minutes. The reaction was carried out. After the enzymatic reaction, 60 μL of trichloroacetic acid was added to a final concentration of 4% to stop the reaction. After the reaction was stopped, the precipitate was removed by centrifugation to prepare the product 23.

（モリブデンブルー法によるピロリン酸の測定：本発明方法の工程（ＩＩ））
比較例１、２及び実施例１６、１７で得られた比較品１、２及び実施品２２、２３のピロリン酸を実施例５に記載のモリブデンブルー法により測定した。その結果、図９に示すように本発明の方法では、添加したアミノ酸が全て反応に使用された場合に産生されると推測されるピロリン酸量である理論値より多くのピロリン酸が産生されていた。一方、比較品の産生したピロリン酸は、理論値以下であった。(Measurement of Pyrophosphoric Acid by Molybdenum Blue Method: Step (II) of the Method of the Present Invention)
The pyrophosphoric acids of Comparative Products 1, 2 and 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 pyrophosphoric acid than the theoretical value, which is the amount of pyrophosphoric acid estimated to be produced when all the added amino acids are used in the reaction, is produced. It was. On the other hand, the pyrophosphate produced by the comparative product was below the theoretical value.

（モリブデンブルー法によるピロリン酸測定におけるアミノ酸検量線）
２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｎＣｌ２、０、又は３０、又は５０、又は７０、又は１００μＭ Ｌ-オルニチン、５．０μＭ Ｐｌｓ−ｓｅ−Ａ（放線菌由来）を含む反応溶液１５０μＬを調製し、４０℃、６０分間反応させた。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、上清中のピロリン酸を実施例５に記載のモリブデンブルー法により測定した。その結果、図１０に示すように、添加したアミノ酸が全て反応に使用された場合に産生されると推測されるピロリン酸量の理論値より多くのピロリン酸が産生されていた。また、０〜１００μＭ のアミノ酸濃度範囲においてアミノ酸濃度とピロリン酸量に相関関係(Ｒ＝０．９９)が認められ、Ｌ-オルニチンの定量が可能であることが示された。(Amino acid calibration curve in measurement of pyrophosphate by molybdenum blue method)
Reactions involving 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 (derived from actinomycetes) 150 μL of the 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%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation, and the pyrophosphoric acid in the supernatant was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 10, more pyrophosphoric acid was produced than the theoretical value of the amount of pyrophosphoric acid estimated to be produced when all the added amino acids were used in the reaction. In addition, 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.

２００ｍＭ ＨＥＰＥＳ−ＫＯＨ（ｐＨ８．０）、４０ｍＭ ＡＴＰ、４０ｍＭ ＭｎＣｌ２、０、又は３０、又は５０、又は７０、又は１００μＭ Ｌ-リジン、５．０μＭ Ｐｌｓ−ｃｇ−Ａ（放線菌由来）を含む反応溶液１５０μＬを調製し、３０℃、６０分間反応させた。反応後、トリクロロ酢酸を終濃度４％となるように３０μＬ添加し、反応を停止した。反応停止後、遠心分離で沈殿を除去し、上清中のピロリン酸を実施例５に記載のモリブデンブルー法により測定した。その結果、図１０示すように添加したアミノ酸が全て反応に使用された場合に産生されると推測されるピロリン酸量の理論値より多くのピロリン酸が産生されていた。また、０〜１００μＭ のアミノ酸濃度範囲においてアミノ酸濃度とピロリン酸量に相関関係(Ｒ＝０．９７)が認められ、Ｌ-リジンの定量が可能であることが示された。Reactions involving 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 (derived from actinomycetes) 150 μL of the 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%, and the reaction was stopped. After the reaction was stopped, the precipitate was removed by centrifugation, and the pyrophosphoric acid in the supernatant was measured by the molybdenum blue method described in Example 5. As a result, as shown in FIG. 10, more pyrophosphoric acid was produced than the theoretical value of the amount of pyrophosphoric acid estimated to be produced when all the added amino acids were used in the reaction. In addition, 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.

以上の結果から、本発明方法におけるＮＲＰＳ反応では、ＮＲＰＳ及びアミノ酸を繰り返し反応に使用することで、産生されるピロリン酸を増幅できることが示された。実施例２〜５に示されるように、Ｌ-オルニチン、Ｌ-リジン及びＤ-リジンの様々なアミノ酸のいずれにおいても当該反応で産生されるピロリン酸をＮＲＰＳ及びアミノ酸のモル数より多く産生させることができた。実施例６〜９に示されるように、ＡＴＰや酵素濃度、実施例１０〜１７に示されるように、二価陽イオンの種類や濃度、反応温度及び反応ｐＨによってもピロリン酸の産生量は変化することが分かった。また、比較例１、２及び実施例１８、１９、２０に示されるように、従来のＮＲＰＳ反応では、産生されるピロリン酸量が、アミノ酸分子数以下であったが、本発明の方法では、アミノ酸分子数以上のピロリン酸が産生されることが分かった。さらに、実施例２１、２２に示されるように、アミノ酸濃度に依存して直線的にピロリン酸が増加する、即ちアミノ酸濃度とピロリン酸量に相関関係があることが分かり、本発明のＮＲＰＳ反応により産生したピロリン酸について、簡便な方法であるモリブデンブルー法による各種アミノ酸の定量が可能であることが確認された。 From the above results, it was shown that in the NRPS reaction in the method of the present invention, the produced pyrophosphoric acid can be amplified by repeatedly using NRPS and amino acids in the reaction. As shown in Examples 2 to 5, in any of the various amino acids of L-ornithine, L-lysine and D-lysine, pyrophosphate produced in the reaction is produced in a larger number than the number of moles of NRPS and amino acids. Was made. As shown in Examples 6 to 9, the amount of pyrophosphoric acid produced also changes depending on the ATP and enzyme concentration, and as shown in Examples 10 to 17, the type and concentration of divalent cations, the reaction temperature and the reaction pH. I found out that Further, as shown in Comparative Examples 1 and 2 and Examples 18, 19 and 20, the amount of pyrophosphate produced in the conventional NRPS reaction was less than or equal to the number of amino acid molecules, but in the method of the present invention, It was found that pyrophosphates with more than the number of amino acid molecules were produced. 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, and the NRPS reaction of the present invention shows that there is a correlation. It was confirmed that various amino acids of the produced pyrophosphate can be quantified by the molybdenum blue method, which is a simple method.

従来のＡＡＲＳを用いたアミノ酸定量法では、タンパク質性アミノ酸（タンパク質を構成する２０種のＬ型のアミノ酸）に対する特異的な２０種類のＡＡＲＳが存在するが、オルニチンなどの２０種類のアミノ酸以外に反応するＡＡＲＳは存在しないため、測定対象のアミノ酸の制限があった。これに対して、ＮＲＰＳは、上記２０種類のアミノ酸に特異的に作用すると共に、異性体であるＤ型のアミノ酸、さらにはメチル化、アシル化、グリコシル化などの修飾アミノ酸、及びオルニチン等の非タンパク質性アミノ酸にも特異的に作用することから、様々なアミノ酸の定量を可能とする。また、本発明方法に於いて、形成されたアミノアシルＡＭＰ-ＮＲＰＳ複合体からＮＲＰＳ及びアミノ酸を遊離し、再度それらをアミノアシルＡＭＰ-ＮＲＰＳ複合体の形成に繰り返し利用することによって、試料中に少ないアミノ酸しか含まれていない場合であっても、多量のピロリン酸や水素イオンを産生させることができるため、多段階酵素反応を用いた蛍光法などによる高感度分析は不必要である。従って、本発明によって、ＮＲＰＳを用いてタンパク質性アミノ酸、また、Ｄ型アミノ酸、修飾アミノ酸及びオルニチン等の非タンパク質性アミノ酸を特異的且つ簡便、高感度に定量する方法及びアミノ酸定量用キットを提供することが可能となった。 In the conventional amino acid quantification method using AARS, there are 20 types of AARS specific to proteinogenic amino acids (20 types of L-type amino acids constituting proteins), but reactions other than 20 types of amino acids such as ornithine Since there is no AARS to be used, there are restrictions on the amino acids to be measured. On the other hand, NRPS acts specifically on the above 20 types of amino acids, and is an isomer D-type amino acid, modified amino acids such as methylation, acylation, and glycosylation, and non-ornithine and the like. Since it acts specifically on proteinogenic amino acids, it enables the quantification of various amino acids. Further, in the method of the present invention, NRPS and amino acids are released from the formed aminoacyl AMP-NRPS complex, and they are repeatedly used for the formation of the aminoacyl AMP-NRPS complex, whereby only a small amount of amino acids are contained in the sample. Even if it is not contained, a large amount of pyrophosphate and hydrogen ions can be produced, so that high-sensitivity analysis by a fluorescence method using a multi-step enzymatic reaction is unnecessary. Therefore, the present invention provides a method for specifically, easily, and highly sensitively quantifying proteinogenic amino acids and non-proteinogenic amino acids such as D-type amino acids, modified amino acids, and ornithine using NRPS, and a kit for quantifying amino acids. It became possible.

実施例１で調製した各ＮＲＰＳは具体的には以下の通り取得した。
目的アミノ酸の活性化を触媒すると予想されるＮＲＰＳについて、推定されるアミノ酸配列をＰｆａｍ ｓｅａｒｃｈ（http://pfam.xfam.org/search#tabview=tab1）にてドメイン検索を行った。その結果から、ペプチド結合形成に必要な各ＮＲＰＳドメイン（Ａドメイン、Ｔドメインなど）に高く保存されている領域を推定し、下記、※１と※２において配列を決定した。
※１. Ａドメインに高く保存されている配列
ＡＭＰ-ｂｉｎｄｉｎｇ ｄｏｍａｉｎ（下線部)
ＡＭＰ-ｂｉｎｄｉｎｇ ｅｎｚｙｍｅ Ｃ-ｔｅｒｍｉｎａｌ ｄｏｍａｉｎ（二重線部）
※２. Ｔドメインに高く保存されている配列
ＬＧＧｘｘＳＬｘＡモチーフ（４´-ｐｈｏｓｐｈｏｐａｎｔｅｔｈｅｉｎｅ ａｔｔａｃｈｍｅｎｔ ｓｉｔｅ）（四角枠内）を中心にもつＰＰ-ｂｉｎｄｉｎｇ ｄｏｍａｉｎSpecifically, each NRPS prepared in Example 1 was obtained as follows.
For NRPS, which is expected to catalyze the activation of the target amino acid, a domain search was performed on the estimated amino acid sequence using Pfam search (http://pfam.xfam.org/search#tabview=tab1). From the results, the regions highly conserved in each NRPS domain (A domain, T domain, etc.) required for peptide bond formation were estimated, and the sequences were determined in * 1 and * 2 below.
* 1. Sequence AMP-binding domain (underlined) that is highly conserved in the A domain
AMP-binding enzyme C-terminal domine (double line part)
* 2. PP-binding domain centered on the sequence LGGxxSLxA motif (4'-phospophantetheine atachment site) (inside the square frame) that is highly conserved in the T domain.

各ＮＲＰＳのアミノ酸配列は以下の通りである。
（１）Ｐｌｓ-ｃｇ-Ａ-ｄｏｍａｉｎ
菌株名：Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍ ＮＢＲＣ１２１６９
アミノ酸配列（大腸菌発現用プラスミドにクローニングした領域）：
［配列番号１］

Figure 2019065211
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］
Figure 2019065211
The amino acid sequence of each NRPS is 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]
Figure 2019065211
Amino acid sequence (recombinant enzyme actually used for activity measurement):
[SEQ ID NO: 2]
Figure 2019065211

（２）Ｐｌｓ-ｃｇ-ＡＴ-ｄｏｍａｉｎ
菌株名：Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍ ＮＢＲＣ１２１６９
アミノ酸配列（大腸菌発現用プラスミドにクローニングした領域）：
［配列番号３］

Figure 2019065211
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］
Figure 2019065211
(2) Pls-cg-AT-domain
Strain name: Corynebacterium glutamicum NBRC12169
Amino acid sequence (region cloned into E. coli expression plasmid):
[SEQ ID NO: 3]
Figure 2019065211
Amino acid sequence (recombinant enzyme actually used for activity measurement):
[SEQ ID NO: 4]
Figure 2019065211

（３）Ｐｌｓ-ｓｅ-Ａ-ｄｏｍａｉｎ
菌株名：Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｅｒｙｔｈｒａｅａ ＮＢＲＣ１３４２６
アミノ酸配列（大腸菌発現用プラスミドにクローニングした領域）：
［配列番号５］

Figure 2019065211
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］
Figure 2019065211
(3) Pls-se-A-domain
Strain name: Saccharoporyspora erythraea NBRC13426
Amino acid sequence (region cloned into E. coli expression plasmid):
[SEQ ID NO: 5]
Figure 2019065211
Amino acid sequence (recombinant enzyme actually used for activity measurement):
[SEQ ID NO: 6]
Figure 2019065211

Claims (7)

［１］以下の各工程を含む工程（Ｉ）：
（工程Ｉ−１）二価陽イオンの存在下、試料中のアミノ酸（ＡＡ）、該ＡＡに対応する非リボソーム型ペプチド合成酵素（ＮＲＰＳ）、及び、アデノシン三リン酸（ＡＴＰ）を反応させて、アミノアシルアデニル酸（アミノアシルＡＭＰ）とＮＲＰＳから成る複合体（アミノアシルＡＭＰ-ＮＲＰＳ複合体）を形成させる反応（反応１）を含む工程；
（工程Ｉ−２）反応１及び／又は反応３で形成されたアミノアシルＡＭＰ-ＮＲＰＳ複合体にアミノ酸再生試薬が作用して、該複合体からＮＲＰＳ及びＡＡが遊離する反応（反応２）を含む工程；
（工程Ｉ−３）反応２で遊離されたＡＡ及び/又はＮＲＰＳを反応１において再利用することによってアミノアシルＡＭＰ-ＮＲＰＳ複合体反応を形成させる反応（反応３）を含む工程；及び、
（工程Ｉ−４）工程Ｉ−２及び工程Ｉ−３を繰り返す工程、並びに、
工程（Ｉ）で生じた反応産生物の量を測定し、該反応産生物の測定量に基づきアミノ酸の量を決定することを含む工程（ＩＩ）、
を含み、前記ＮＲＰＳは少なくともＡドメインを含む、試料中のアミノ酸定量方法。[1] Step (I) including each of the following steps:
(Step I-1) In the presence of divalent cations, the amino acid (AA) in the sample, the nonribosomal peptide synthase (NRPS) corresponding to the AA, and adenosine triphosphate (ATP) are reacted. , A step comprising a reaction (reaction 1) to form a complex (aminoacyl AMP-NRPS complex) consisting of aminoacyl-adenylic acid (aminoacyl AMP) and NRPS;
(Step I-2) A step including a reaction (Reaction 2) in which an amino acid regeneration reagent acts on the aminoacyl AMP-NRPS complex formed in Reaction 1 and / or Reaction 3 to release NRPS and AA from the complex. ;
(Step I-3) A step including a reaction (reaction 3) in which the AA and / or NRPS released in the reaction 2 is reused in the reaction 1 to form an aminoacyl AMP-NRPS complex reaction;
(Step I-4) A step of repeating steps I-2 and I-3, and
Step (II), which comprises measuring the amount of reaction product produced in step (I) and determining the amount of amino acid based on the measured amount of the reaction product.
A method for quantifying amino acids in a sample, wherein the NRPS contains at least the A domain. ＮＲＰＳがＡドメインから構成されるもの、並びに/又は、Ａドメイン及びＴドメインから構成されるものである、請求項１に記載のアミノ酸定量方法。 The amino acid quantification method according to claim 1, wherein the NRPS is composed of an A domain and / or an A domain and a T domain. 工程（Ｉ）で用いるアミノ酸再生試薬が、ヌクレオチド及び／又はアルカリ性化合物であることを特徴とする、［１］に記載のアミノ酸定量方法。 The amino acid quantification method according to [1], wherein the amino acid regeneration reagent used in the step (I) is a nucleotide and / or an alkaline compound. 吸光度法により吸光度変化を測定することによって、工程（Ｉ）で生じた反応産生物の量を測定する、請求項１〜３の何れか一項に記載のアミノ酸定量方法。 The amino acid quantification method according to any one of claims 1 to 3, wherein the amount of the reaction product produced in the step (I) is measured by measuring the change in absorbance by the absorbance method. 工程（Ｉ）で生じる反応産生物として、ピロリン酸又は水素イオンの少なくとも何れか１つを測定する、請求項１〜４の何れか一項に記載のアミノ酸定量方法。 The amino acid quantification method according to any one of claims 1 to 4, wherein at least one of pyrophosphate or hydrogen ion is measured as the reaction product produced in the step (I). 工程（Ｉ）で生じた反応産生物のモル数が試料中のＮＲＰＳ及び／又はアミノ酸のモル数より多いことを特徴とする、請求項１〜５のいずれか一項に記載のアミノ酸定量方法。 The amino acid quantification method according to any one of claims 1 to 5, wherein the number of moles of the reaction product produced in the step (I) is larger than the number of moles of NRPS and / or amino acids in the sample. 請求項１〜６に記載のアミノ酸定量法を実施するためのアミノ酸定量用キットであって、ＡＴＰ、ヌクレオチド及び該アミノ酸に対応するＮＲＰＳを含む、アミノ酸定量用キット。 An amino acid quantification kit for carrying out the amino acid quantification method according to claims 1 to 6, which comprises ATP, nucleotides and NRPS corresponding to the amino acid.
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