JP2004283121A - Isoamylase derived from porphyridium belonging to rhodophyta - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To analyze the starch structure of Rhodophyta and separate and identify the synthetase of the starch. <P>SOLUTION: The invention relates to an isoamylase derived from Porphyridium belonging to Rhodophyta. The invention further relates to a gene encoding the isoamylase, a vector containing the gene and a transformant containing the vector. The starch of Rhodophyta has a special structure different from the starch of other organisms, and is useful as a new starch for food and industrial use and the synthetase is useful as an enzyme for the production of starch having a new structure. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００１９】
次に、ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）とイネ（Ｆｕｊｉｈｉｋａｒｉ）のゲル濾過クロマトグラフィで得られたフラクションのアミロペクチンとアミロースの画分の鎖長分布を解析した。両者の鎖長分布を図１２に示す。図１２の（ａ）（上段）はアミロペクチン画分の比較であり、（ｂ）（下段）はアミロース画分の比較である。図１２の灰色の棒はイネ（Ｆｕｊｉｈｉｋａｒｉ）を示し、黒色の棒は紅藻ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）を示す。図１２（ａ）及び（ｂ）の横軸は鎖長（グルコースの数（ＤＰ））を示し、縦軸は各鎖長の量の全鎖長の合計の量に対する比率（％）を示す。
ポルフィリディウム・プルプレウム（Ｐ． ｐｕｒｐｕｒｅｕｍ）ではアミロペクチン画分と比較するとアミロース画分の方が短差の量が若干多いものの、両画分は非常によく似た鎖長分布を示した。アミロース画分へのアミロペクチンの混在によってアミロースだけの鎖長分布が得られなかったためと思われる。この点について、Ｆｕｊｉｈｉｋａｒｉでも同様のことがいえた。
また、ポルフィリディウム・プルプレウム（Ｐ． ｐｕｒｐｕｒｅｕｍ）のアミロペクチンはＦｕｊｉｈｉｋａｒｉに比べＤＰ８以下の短鎖が多いが、ＤＰ１０−２５付近までの分布は少なかった。また、ＤＰ４５付近にも違いが見られ、Ｆｕｊｉｈｉｋａｒｉではなだらかなピークが見られるのに対し、ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）ではそのピークは見られなかった。
【００２０】
以上のことから、紅藻ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）のデンプンはイネのものと比べると短鎖が多く、クラスター構造を形成するために必要と考えられているＤＰ４５付近の長鎖のピークが低く、長鎖のものが少ないことがわかる。ラン藻類は、主にＤＰ１０以下の短鎖が非常に多く、クラスター構造を形成するために必要だと思われるＤＰ４５付近にピークをもた無いことが知られているが、緑藻類は短鎖の分布にわずかな違いがあるが、高等植物のイネと類似した鎖長分布を示し、ＤＰ４５付近のピークも見られることからクラスター構造を形成していることが示された。イネと緑藻類では、短鎖にはわずかの違いがみられるが、ＤＰ４５付近の長鎖のピークは共通して観察されているため、緑藻のほうが紅藻よりも高等植物に近いデンプン構造を形成していると考えられる。ラン藻類はフィトグリコーゲンを合成するため、鎖長分析ではＤＰ３−８付近までの短鎖が非常に多く、ＤＰ３０以降の長鎖が無いものが多い。
したがって、ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）はラン藻類と比べると短鎖は少ないがＤＰ３０以降に長鎖が見られるということになる。これらのことから、ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）のデンプンはラン藻類とイネの中間型であると考えられる。構造的にはイネのアミロペクチンのクラスター構造ほどしっかりした構造は形成できてはいないが、ラン藻類のフィトグリコーゲンほど乱雑に分枝してはいないと考えられる。一方、ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）とは異なる鎖長分布を示した紅藻シアニディウム・カルダリウム（Ｃ．ｃａｌｄａｒｉｕｍ）とガルディエリア・サルフラリア（Ｇ．ｓｕｌｐｈｕｒａｒｉａ）は非常に短鎖が多く、ラン藻類に近い鎖長分布であると考えられる。
大型藻であるＧｅｌｉｄｉｕｍ（テングサ）ではＤＰ１０−１５付近の側鎖が少ないが、それ以外はポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）の場合の鎖長分析と非常に似ていることからＧｅｌｉｄｉｕｍのデンプンはシアニディウム・カルダリウム（Ｃ．ｃａｌｄａｒｉｕｍ）やガルディエリア・サルフラリア（Ｇ．ｓｕｌｐｈｕｒａｒｉａ）のものよりもポルフィリディウム・プルプレウム（Ｐ． ｐｕｒｐｕｒｅｕｍ）のものと似たような構造を含んでいると考えられる。
【００２１】
このように、紅藻類でもアミロースをもつものともたないものの存在が明らかになった。特にガルディエリア・サルフラリア（Ｇ．ｓｕｌｐｈｕｒａｒｉａ）ではフェノール硫酸法では還元糖の存在が確認されたのにもかかわらず、ヨウ素デンプン反応での呈色は得られなかった。また、ポルフィリディウム・プルプレウム（Ｐ． ｐｕｒｐｕｒｅｕｍ）のデンプンのゲル濾過クロマトグラフィの溶出位置を比べると、ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）では４０ｍｌ付近から溶出されているのに対し、ガルディエリア・サルフラリア（Ｇ．ｓｕｌｐｈｕｒａｒｉａ）では８０ｍｌ付近から溶出されていた。これらのことからガルディエリア・サルフラリア（Ｇ．ｓｕｌｐｈｕｒａｒｉａ）のポリグリカンは一つの構成体からなっており、ヨウ素デンプン反応では染色されにくく、ポルフィリディウム・プルプレウム（Ｐ． ｐｕｒｐｕｒｅｕｍ）のアミロペクチン画分よりも小さい分子量をもつことがわかる。その構成体は不完全なアミロペクチン構造かフィトグリコーゲン様の構造であると考えられ、鎖長分析の結果も加えるとフィトグリコーゲン様の構造をしていると考えられる。
また、紅藻類、緑藻類ともにイネに比べると圧倒的にアミロースが少ないことがわかった。
【００２２】
高等植物ではイソアミラーゼ（ＩＳＡ）がデンプンのクラスター構造の形成に必須であると考えている。そこで、デンプンのクラスター構造の存在が確認された紅藻ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）においてＩＳＡの存在を検討した。
ヨウ素デンプン反応を利用した活性染色（Ｎａｔｉｖｅ ＰＡＧＥ）により、ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）のＩＳＡの活性を試験した結果を図１３に図面に代わる写真で示す。図１３レーン１及び２は、マーカーとしてのイネの登熟種子抽出液であり、レーン３は紅藻ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）の細胞をガラスビーズで破砕した破砕液であり、レーン４はその１８，０００×ｇでの遠心分離の上澄液である。図１３の左側にはプルラナーゼ（ＰＵＬ）の位置を示し、右側にはイソアミラーゼ（ＩＳＡ）の位置を示している。
この結果、紅藻ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）においてもＩＳＡの存在を確認することができた（図１３のレーン３及び４参照）。このことから、緑色植物のようにポルフィリディウム・プルプレウム（Ｐ． ｐｕｒｐｕｒｅｕｍ）でもＩＳＡがアミロペクチンの合成か分解に関与していることが考えられる。
なお、この活性染色では、ゲル中のアミロペクチンは、ＩＳＡでα−１，６グリコシド結合を切断され分枝が少なくなるとヨウ素に染まりやすくなり青色を呈するが、アミラーゼ（ａｍｙｌａｓｅ）で切断されるとグルコースやマルトースそしてオリゴサッカライドのような糖に分解されるためヨウ素によって染色されなくなる。したがって、図１３で色が白く抜けて見えるバンドがいくつか観察されているがこれはアミラーゼであると考えられる。
【００２３】
紅藻はＵＤＰ−ｇｌｕｃｏｓｅをグルコースドナーとすることや、アミロースを合成するものとしないものとが存在することなど、独特のデンプン代謝系を持っていると考えられる。そこで、デンプン合成関連酵素の中から、前記の実験により確認することができたＩＳＡのｃＤＮＡをまずクローニングしてその塩基配列を決定し、ラン藻、緑藻及び高等植物と比較し、遺伝子レベルでどのような違いがあるかを検討することにした。
【００２４】
ＩＳＡには、ＩＳＡ遺伝子に共通に見られる領域（保存領域）が存在していることが知られている。これを図１４に示す。ここに例示しているのはトウモロコシ（ｍａｉｚｅ ｅｎｄｏｓｐｅｒｍ）、イネ（ｒｉｃｅ ｅｎｄｏｓｐｅｒｍ）、ラン藻（Ｓｙｎｅｃｈｏｃｙｓｔｉｓ）の２種である。いずれも図１４に示した、Ｉ、ＩＩ、ＩＩＩ、及びＩＶの４つの領域において保存領域がある。
そこで、ＩＳＡ遺伝子に共通に見られる領域（保存領域）のアミノ酸配列をもとに作製したプライマーを用いて、紅藻ポルフィリディウム・プルプレウム（Ｐ． ｐｕｒｐｕｒｅｕｍ）のｃＤＮＡをテンプレートにしてＰＣＲを行った。プライマーの種々な組み合わせでＰＣＲを行ったところ、Ｆ１とＲ１のプライマーでＰＣＲを行ったのち、そのＰＣＲ産物をテンプレートとしてＦ１とＲ２のプライマー（図１４の下段に示す。）でネステッド（ｎｅｓｔｅｄ）ＰＣＲを行った場合に、予想される大きさのＰＣＲ産物が得られた。得られたＰＣＲ断片をシークエンスした。得られた塩基配列から予想されるアミノ酸配列は他の生物のＩＳＡのものと相同性を示したため、この部分の塩基配列（ＢｏｘＩ、ＩＩの間の配列）をもとに６８ｍｅｒのプローブを作製した。
【００２５】
このプローブを用いて、約５×１０^５クローンについてスクリーニングしたところ、１次スクリーニングで５個のポジティブクローンが得られ、さらに２次スクリーニングを行ったところ５種類全てについてポジティブクローンが得られた。５個のポジティブクローンのうち、インビボエクサイション（ｉｎ ｖｉｖｏ Ｅｘｃｉｓｉｏｎ）がうまくいったのは２種類で、また、インビボエクサイション（ｉｎ ｖｉｖｏ Ｅｘｃｉｓｉｏｎ）のときの希釈が異なるものを加えると４個であった。プレートから２個ずつとり、計８個をシークエンスにかけた。シークエンスの結果、８クローンのうち同様の配列が得られたものを除き、異なる配列のクローンが３クローン得られた。
得られた３クローンのうち、クローン２はホモロジー検索からシロイヌナズナ（Ａｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａ）のＤｅｇＰタンパク質（ＡＹ０３９５８５−１）と相同性が高かったので、解析を終わらせた。
クローン１とクローン３は上述のＰＣＲ産物の配列に一致する配列を含んでいた。
これらのクローンから解読できた塩基配列を配列表の配列番号１に示し、また、その遺伝子がコードしていると予想されるアミノ酸配列を配列番号２に示す。図１５にアミノ酸の１文字表記によるアミノ酸配列を示す。
【００２６】
クローン１とクローン３の塩基配列の相同性は９９．４％であり、これらのクローンの塩基配列から予想されるアミノ酸配列は他の生物のＩＳＡのものと高い相同性を示した。図１６及び図１７に各種の生物のＩＳＡと本発明のＩＳＡのアミノ酸配列の比較を示す。図１６及び図１７は、上から本発明の紅藻ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）の解読できた部分のアミノ酸配列を示し、その下の段はイネのＩＳＡ（Ｒｉｃｅ−ＩＳＡ）を示し、その下の段はラン藻ｇｌｇＸのＩＳＡ（Ｓｃｙｓｔｉｓ−ｇｌｇＸ）を示し、その下の段はラン藻ｇｌｇＸＸのＩＳＡ（Ｓｃｙｓｔｉｓ−ｇｌｇＸＸ）を示し、その下の段はＰｓｅｕｄｏｍｏｎａｓ ａｍｙｌｏｄｅｒａｍｏｓａのＩＳＡ（Ｐ．ａｍｙｌｏ−ＩＳＡ）を示す。図１７は図１６の続きである。
また、クローン１の配列に他の生物のＩＳＡの配列を加えて作製した系統樹を図１８に示した。図１８の各枝の上の数字はブーツストラップを示す。
【００２７】
本発明は、紅藻の原始紅藻亜綱のチノリモ目（Ｐｏｒｐｈｙｒｉｄｉａｌｅｓ）のチノリモ属（Ｐｏｒｐｈｙｒｉｄｉｕｍ）、より詳細にはプルプレウム種（Ｐｏｒｐｈｙｒｉｄｉｕｍ ｐｕｒｐｕｒｅｕｍ）に由来するイソアミラーゼ、及びそれをコードする遺伝子を提供するものである。本発明は紅藻のイソアミラーゼの存在を確認し、当該酵素をコードする遺伝子をクローニングしたものであり、その塩基配列については全部の解析が完了していないが、本明細書に記載した方法により目的の紅藻の遺伝子を入手でき、常法によりその塩基配列を解読することができる。
本発明のイソアミラーゼは、前記した例ではプルプレウム種（Ｐ． ｐｕｒｐｕｒｅｕｍ）のＲ−１に由来したものが示されているが、これに限定されるものではない。
本発明のイソアミラーゼは、前記してきたように、紅藻に特有なデンプンである、ラン藻類とイネの中間型のような、構造的にはイネのアミロペクチンのクラスター構造ほどしっかりした構造は形成できてはいないが、ラン藻類のフィトグリコーゲンほど乱雑に分枝してはいない構造を有するデンプンを製造に有用なものと考えられる。一般に、クラスター構造がしっかりしていないデンプンは、しっかりしているデンプンに比べて水溶性になり、また糊化温度などの特性も特異的なものとなり、全く新規なデンプンを製造するために重要な酵素であると考えられる。
本発明のイソアミラーゼは、一部のアミノ酸配列が想定されているが（配列番号２参照）、このアミノ酸配列を有するものに限定されるものではなく、紅藻類、好ましくはチノリモ目（Ｐｏｒｐｈｙｒｉｄｉａｌｅｓ）、より好ましくはチノリモ属（Ｐｏｒｐｈｙｒｉｄｉｕｍ）、さらに好ましくはプルプレウム種（Ｐｏｒｐｈｙｒｉｄｉｕｍ ｐｕｒｐｕｒｅｕｍ）に由来するものであり、前記した紅藻類に特有なデンプンを形成できる活性を有するものであればよい。
【００２８】
本発明のイソアミラーゼは、前記した方法により製造することができるが、この方法に限定されるものではなく、本発明により提供される塩基配列を利用してＰＣＲ法などの公知の手法を組み合わせることにより各種の方法で製造することができる。
また、本発明の遺伝子のクローニング方法も前記した手法に限定されるものではなく、公知の各種のクローニング手法を利用することができる。
さらに、本発明のイソアミラーゼの遺伝子は必要により、その上流及び／又は下流側に各種の塩基配列を有する遺伝子を接続して使用することができるが、いずれの場合であっても本発明の遺伝子を含有している限りにおいては本発明の技術的範囲に属するものである。本発明のイソアミラーゼ遺伝子の上流側に接続できる遺伝子の例としては各種の生物から誘導されるプロモーターが挙げられる。プロモーターとしては紅藻ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）のもを使用することもできるが、他の生物のものを組み合わせることもできる。
【００２９】
本発明は、前記した本発明のイソアミラーゼ遺伝子を含有しているベクターを包含している。ベクターとしては、前記の例でしめしてきたようなクローニングベクターでもよく、またプロモーターを結合させた発現ベクターであってもよい。べくたーとしては各種のプラスミドを選定することができる。
また、本発明は、前記してきた本発明の遺伝子を導入した形質転換体を提供する。本発明の遺伝子が導入される宿主細胞としては、特に制限はないがデンプンを生産できる細胞が好ましい。宿主細胞が既にイソアミラーゼの遺伝子を有している場合にはこれをノックアウトしておいてもよいし、そのままの状態で本発明の遺伝子を導入してもよい。このようにして本発明の遺伝子が導入され、発現した細胞は、本発明のイソアミラーゼの活性によりデンプンの枝切りが行われ、紅藻のデンプンに近い構造をしたデンプンの産生が行われるものと考えられる。
【００３０】
本発明は、また、本発明のイソアミラーゼ遺伝子の部分配列からなるオリゴヌクレオチドを提供するものである。本発明のオリゴヌクレオチドは、ＰＣＲにおけるプライマーや、クローニングのプローブや、本発明の遺伝子の存在を検出するためのプローブなどとして有用である。本発明のオリゴヌクレオチドの長さに特に制限はないが、好ましくは１０〜１５０塩基、１５〜１５０塩基、２０〜１５０塩基、１５〜５０塩基などの長さのものを適宜使用することができる。
【００３１】
【実施例】
以下、実施例により本発明をより具体的に説明するが、本発明はこれら実施例により何ら限定されるものではない。
生物材料の、ポルフィリディウム・プルプレウムＲ−１（Ｐｏｒｐｈｙｒｉｄｉｕｍ ｐｕｒｐｕｒｅｕｍ Ｒ−１）（チノリモ）、ガルディエリア・サルフラリアＭ−８（Ｇａｌｄｉｅｒｉａ ｓｕｌｐｈｕｒａｒｉａ Ｍ−８）、及びシアニディウム・カルダリウムＮ−５５１（Ｃｙａｎｉｄｉｕｍ ｃａｌｄａｒｉｕｍ Ｎ−５５１）の３種は東京大学分子細胞学研究所のＩＡＭカルチャーコレクションより入手した。スサビノリ（Ｐｏｒｐｈｙｒａ ｓｐ．）は神戸大学内海城機能教育研究センターより入手した。テングサ（Ｇｅｌｉｄｉｕｍ ｓｐ．）は愛媛の浜から採取したものである。
ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）はＭＫＭ培地で２０℃、２％ＣＯ_２のバブリングで、ガルディエリア・サルフラリア（Ｇ．ｓｕｌｐｈｕｒａｒｉａ）はアレン（Ａｌｌｅｎ）培地で３３℃、空気のバブリングで、シアニディウム・カルダリウム（Ｃ．ｃａｌｄａｒｉｕｍ）はＡ１５Ｌ培地で３３℃、空気のバブリングの条件で培養した。
【００３２】
実施例１ （デンプンの抽出）
（１）小型生物からの抽出
数日間培養したポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）等の細胞を１，７００×ｇ、４℃、１０分間で遠心した。メタノールを加えガラスビーズで１０分間ボルテックスにかけ、細胞を破砕した。１，７００×ｇ、４℃、１０分間で遠心後、沈殿に対し再度メタノールを加え、色素がぬけるまで上記の操作を繰り返した。遠心した沈殿にジメチルスルホキシド（ＤＭＳＯ）を加え、９０℃で１２０分間煮沸した。煮沸後、５０ｍｌのチューブに移し、１，７００×ｇ、４℃、１０分間で遠心し、上清を１Ｌのメディウムビンにいれ、沈殿にもう一度ＤＭＳＯを加え、９０℃で１２０分間煮沸した。煮沸後、もう一度上記の遠心操作を行い、得られた上清を１Ｌのメディウムビンに入れエタノールを８００ｍｌ程加えて、−２０℃で一晩おき、デンプンを析出させた。
【００３３】
（２）大型藻類からの抽出
Ｐｏｒｐｈｙｒａ ｓｐなどの大型藻を乳鉢に入れ、液体窒素で凍らせ乳棒ですりつぶした。ある程度すりつぶした後メタノールを加え撹拝し−２０℃で一晩おいた。次の日２，０００×ｇで１０分間遠心し沈殿を乳鉢にいれ前日と同様の操作を行い細胞をできるだけ破砕した。細胞破砕液を２，０００×ｇで１０分間遠心してメタノ−ルを取り除き、ＤＭＳＯを加えた。後の操作は微細藻類のデンプン抽出と同様に行った。
【００３４】
実施例２ （キャピラリー電気泳動による分析）
デンプンのα−１，６グリコシド結合をイソアミラーゼで完全分解して得たさまざまな長さのα−１，４グルカンの集合体を得た。酵素処理で得られたα−１，４グルカンすべての還元末端に蛍光物質であるＡＴＰＳ（１−ａｍｉｎｏｐｙｒｅｎｅ−３，６，８−ｔｒｉｓｕｌｆｏｎｉｃ ａｃｉｄ ｔｒｉｓｏｄｉｕｍ ｓａｌｔ）を付加し、それらを電気泳動して、４８８ｎｍのレーザー誘導蛍光ディテクターで高感度検出して、定量した。
結果を、図１〜図４にそれぞれ示す。比較としてイネのデンプンの鎖長分布を図５に示す。
【００３５】
実施例３ （デンプンのゲル濾過クロマトグラフィによる構造解析）
（１）サンプルの調製
微細藻類のデンプンは、エタノールを１ｍｌ加え懸濁し、１０，０００×ｇ、４℃、１０分で遠心し上清を除き、アスピレーターで乾燥させた。
イネ（Ｆｕｊｉｈｉｋａｒｉ）のデンプンは、胚乳を乳鉢に入れ乳棒ですりつぶしてエタノールを加え懸濁し、１８，０００×ｇで５分間遠心した。沈澱をアスピレーターで乾燥させデンプンを得た。
デンプン２０ｍｇに１．６ｍｌの蒸留水を加えて懸濁し、０．４ｍｌの５Ｎ ＮａＯＨを加えて混合した。４７℃の湯浴に３０分間放置し、デンプンを糊化させた。２．０ｍｌの蒸留水を加えて混合し、２，０００×ｇで５分間、室温で遠心分離し、上清３．０ｍｌを別の試験管に移しゲル濾過サンプルとした。
（２）ゲル濾過クロマトグラフィ
ゲル濾過クロマトグラフィによる構造解析はナカムラらの方法（Ｎａｋａｍｕｒａ， Ｙ．， ｅｔ ａｌ．， Ｐｌａｎｔ Ｊ．， （１９９７） ， １２（１）， １４３−１５３）に準じて行った。フラクションコレクタ一（ＡＴＴＯ）に分取用試験管（６ｍｌ）を６０本セットした。流速は約０．１４ｍｌ／分で、試験管１本あたり約４ｍｌずつ分画分取した。
得られたポルフィリディウム・プルプレウム（Ｐ． ｐｕｒｐｕｒｅｕｍ）のゲル濾過クロマトグラフィの各画分のヨウ素デンプン反応による呈色反応の結果を図６に示す。
また、図７〜図１１にそれぞれのゲル濾過クロマトグラフィの結果を示す。
【００３６】
実施例４ （ゲル濾過における各画分の鎖長分析）
ゲル濾過クロマトグラフィによって得られたフラクションのうち、λｍａｘの測定よりアミロぺクチンとアミロースと推定されるフラクションをそれぞれメディウムビンに集めエタノールを加え、−２０℃で一晩おいて、デンプンを析出させた。遠心によりデンプンを回収し、それぞれを前記したキャピラリー電気泳動法により鎖長の分析を行った。
結果を、図１２に示す。
【００３７】
実施例５ （デンプン合成・分解関連酵素の活性染色（Ｎａｔｉｖｅ ＰＡＧＥ））
デンプン合成・分解関連酵素活性を調べるために、ネイティブのゲルを用いて電気泳動をし、活性染色を行った。
アクリルアミドゲルを次のようにして調製した。
ポテトアミロぺクチン（２４ｍｇ）をビーカーに測り取り３．３ｍｌの蒸留水を加えて混合しラップをかぶせてスターラーで回しながら６０℃で透明になるまで溶かした。３０％アクリルアミド、０．８％ ビス−アクリルアミドと１．５Ｍトリス−ＨＣｌ（ｐＨ ８．８）、０．２％ ＴＥＭＥＤをそれぞれ１．６５ｍｌ、アミロペクチン溶液３．３ｍｌを混合し１０％ ＡＭＰＳ（過硫酸アンモニウム）を３０μｌ加え、ゲル板に流し込んだ。ブタノールを重層し、約３０分放置した。３０分後ブタノ−ルを取り除き蒸留水でゲル上部を洗浄した。３０％アクリルアミド、０．８％ビス−アクリルアミド、１．０Ｍトリス−ＨＣｌ （ｐＨ ６．８）、０．２％ ＴＥＭＥＤをそれぞれ０．３ｍｌ、蒸留水を１．８ｍｌ加え混合しＡＰＳを１６μｌ加えゲル板に流し込み、１０または１２レーンのコームをさし、２０分放置した。
イネ（登熟）の籾と胚を取り除き、エッペンドルフチューブに入れて、胚乳の重量を測定した。胚乳に胚乳重量の３−４倍量の細胞破砕用バッファー（Ｇｒｉｎｄ Ｓｏｌｕｔｉｏｎ（以下、Ｇｓと略す）（５０ｍＭイミダゾール−ＨＣｌ（ｐＨ ７．４）、８ｍＭ ＭｇＣｌ_２、１２．５％グリセロール、５００ｍＭ ２−メルカプトエタノール）加え、プラスチックペッスルですりつぶした。１８，０００×ｇ、４℃で２０分間遠心分離し、上清を別のチューブに入れれた。この上清とサンプルバッファーが２：１になるように混合し、泳動マーカーとした。
サンプルの細胞は、１，７００×ｇ、１０分で細胞を集めた。集めた細胞を細胞破砕用バッファー（Ｇｓ）で懸濁した。ガラスビーズの入った試験管に懸濁した細胞液を入れ、１０分間ボルテックスをかけ細胞を破壊し、１８，０００×ｇ、５分遠心し上清を泳動サンプルとした。
【００３８】
前記で調製したマーカー及びサンプルを用いて、先に調製したアクリルアミドゲルで電気泳動した。
泳動後、ゲルをゲル板からはずし反応液の入った容器に入れ、５分間、３０℃で振盪した。同じ操作を２回繰り返した後、ゲルを反応液に浸し、１２０分間、３０℃でインキュベートした。反応液を捨て、ヨウ素溶液を入れ、１５分ほど振盪してゲルを染色した。染色後イルミネーターで観察を行った。
結果を図１３に示す。
【００３９】
実施例６ ポルフィリディウム・プルプレウムＲ−１（Ｐ． ｐｕｒｐｕｒｅｕｍ Ｒ−１）の
イソアミラーゼ（ＩＳＡ）ｃＤＮＡクローンの単離
（１） ＩＳＡプローブの作製
ＩＳＡ遺伝子に共通に見られる領域（保存領域）（図１４参照）のアミノ酸配列をもとに作製したプライマー（フォーワードプライマー，Ｆ１、及びＦ２；リバースプライマー，Ｒ１、及びＲ２）を用いて、ポルフィリディウム・プルプレウムＲ−１（Ｐ． ｐｕｒｐｕｒｅｕｍ）のｃＤＮＡをテンプレートにしてＰＣＲを行い、得られたＰＣＲ断片をシークエンスした。得られた塩基配列から予想されるアミノ酸配列は他の生物のＩＳＡのものと相同性を示したため、この部分の塩基配列（ＢｏｘＩ、ＩＩの間の配列）をもとに６８ｍｅｒのプローブを作製した。
【００４０】
（２） １次スクリーニング
ＬＢ培地で終夜培養した大腸菌（ＸＬ１−ｂｌｕｅ ｑＬＩ−ｂｌｕｅ ＭＲＦ’ ｓｔｒａｉｎ）の濃度（ＯＤ_６００）を測定し、１，５００×ｇ、１０分間遠心し、沈澱を大腸菌の濃度がＯＤ_６００＝０．５になるよう１０ｍＭ ＭｇＳＯ_４で懸濁した。この大腸菌懸濁液１．５ｍｌにファージ液８μｌ（１０，０００クローン）を加え、３７℃で１５分間インキュベートした。
インキュベート後ファージに感染させた大腸菌６００μｌをソフトアガーに加え、３７℃のインキュベーターで保温しておいたプレート（１５０ｍＭ）培地に素早く広げた。１５分間室温で静置しソフトを固まらせた。固まったプレート培地を逆さにして３７℃のインキュベーターで６〜８時間程静置し、その後４℃で保存した。
メンブレン（Ｈｙｂｏｎｄ−Ｎ^＋、Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ）をプレート培地の上に気泡が入らないように静かにのせ、１分間静置した。墨汁をつけた注射針で３箇所突き刺しマークをつけた。ピンセットでメンブレンを剥がした。この操作をプレート１枚につきメンブレン２枚ずつ行った。メンブレンのプレート培地と接していた面を上にして変性溶液を浸み込ませたろ紙の上に７分間静置した。静置後、メンブレンを乾いたろ紙の上に置き、中和液を浸み込ませたろ紙の上に３分間静置した。この操作を２回繰り返した。メンブレンを２×ＳＳＣバッファーに浸し、１分間程度洗浄した。メンブレンを乾いたろ紙の上に置き乾燥させ、８０℃で２時間加熱した。
前記で調製したプローブ（放射性元素で標識）を用いて、プレハイブリダイゼーションとハイブリダイゼーションを行った。プレハイブリダイゼーションとハイプリダイゼーションにはラピッド−ハイブバッファー（Ｒａｐｉｄ−ｈｙｂ ｂｕｆｆｅｒ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ））を用いた。４２℃で温めておいたＲａｐｉｄ−ｈｙｂ ｂｕｆｆｅｒにメンブレンを浸し４２℃、３０分間振盪しプレハイブリダイゼーションを行わせた。
Ｘ線フィルムを現像して、ポジティブプラークの有無を調べた。
【００４１】
（３） ２次スクリーニング
得られたポジティブプラークをプレートから切り出し、５００μｌのＳＭバッファー（１Ｌ中、ＮａＣｌ ５．８ｇ、ＭｇＳＯ_４・７Ｈ_２Ｏ ２．０ｇ、１Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ ７．５） ５０．０ｍｌ、２％ｇｅｌａｔｉｎ ５．０ｍｌ）が入ったエッペンドルフチューブに入れクロロホルムを一滴加えた。ボルテックス後４℃で保存した。
１次スクリーニングと同様にＬＢ培地で大腸菌を培養し、１，５００×ｇ、１０分の条件で遠心し、沈澱にＯＤ_６００＝０．５になるように１０ｍＭ ＭｇＳＯ_４を加え懸濁した。ＯＤ_６００＝０．５の宿主細胞２００μｌに対して、ファージ原液をＳＭバッファーで１／１００、１／１，０００あるいは１／１０，０００に希釈したものを１μｌずつ加え、３７℃で１５分間インキュベートした。インキュベート後ファージに感染させた大腸菌を３ｍｌのトップに加え、ＮＺＹプレート（９０ｍＭ）に広げた。室温で１０分間トップを固まらせ、プレートを逆さにして３７℃で一晩保温しプラークを得た。１次スクリーニングと同様に１枚のプレートにつき２枚ずつハイブリダイゼーション用メンブレンを作製した。
この後の実験操作は現像まで１次スクリーニングと同様の方法で行った。
【００４２】
（４） インビボエクサイション（ｉｎ ｖｉｖｏ Ｅｘｃｉｓｉｏｎ）
ポジティブプラークをプレートから切り出し、５００μｌのＳＭバッファーが入ったエッベンドルフチューブに入れクロロホルムを一滴加えた。ボルテックス後４℃で保存した。ＸＬ１−ｂｌｕｅ ＭＲＦ’株とＳＯＬＲ株を、ＬＢ培地に０．２％マルトースと１０ｍＭ ＭｇＳＯ_４を加えた培地で、それぞれ３０℃で終夜培養を行った。１，０００×ｇ、１０分間遠心しＯＤ_６００＝１０になるように１０ｍＭ ＭｇＳＯ_４を加え懸濁した。ＸＬ１−ｂｌｕｅ ＭＲＦ’株（ＯＤ_６００＝１．０）２００μｌ、ファージストック２５０μｌ及びＥＸ Ａｓｓｉｓｔ ｈｅｌｐｅｒ ｐｈａｇｅ １μｌ をチューブに入れ、３７℃で１５分間インキュベートした。３ｍｌのＬＢ培地を加え２〜３時間、３７℃で振盪培養を行った。培養後、６５〜７０℃で２０分間加熱μｌ，０００×ｇで１５分間遠心した。その上清をデカンテーションで新しいチューブに移した。ストックとする場合は４℃で保存した。プレーティングを行う場合は、ＳＯＬＲ ｃｅｌｌｓ（ＯＤ_６００＝１．０）２００μｌにファージの上清を１００μｌもしくは１０μｌ加え、３７℃で１５分間インキュベートした。それぞれの細胞混合物２００μｌをＬＢ−アムピシリンアガープレートにまき３７℃で終夜培養を行った。
【００４３】
（４） プラスミドの抽出
前記の（３）で得られたコロニーを楊枝でつつき２×ＹＴ培地とアンピシリンの入った培地に植え、３７℃で終夜培養を行った。３，０００×ｇ、１分間遠心し、上澄みを捨てた。溶液Ｉ（５０ｍＭ グルコース、２５ｍＭ トリス−ＨＣｌ（ｐＨ ８．０）、１０ｍＭ ＥＤＴＡ（ｐＨ ８．０））を２００μｌ加えピペッティングで混ぜ、溶液ＩＩ（０．２Ｎ ＮａＯＨ、１％ ＳＤＳ）を３００μｌ加えチューブを倒置して混ぜ、氷上で５分間静置した。溶液ＩＩＩ（３Ｍ ｐｏｔａｓｓｉｕｍ ａｃｅｔａｔｅ（ｐＨ ４．８））を３００μｌ加えチューブを倒置して混ぜ氷上に５分間静置し、３，０００×ｇ、室温、１０分間遠心して上澄みを新しいチューブに移した。上澄みに１０ｍｇ／ｍｌ ＲＮａｓｅ Ａを２μｌ加え３７℃で２０分間静置した。クロロホルムを４００μｌ加え手で転がすように３０秒境拝した後、１分間遠心し、水層（上層）を新しいチューブに移した。この操作を２回行った。水層に６００μｌの２−プロパノールを加えて１８，０００×ｇ、室温で１０分間遠心し、上清を捨て沈澱に７０％エタノール５００μｌを加えた。もう一度同様に遠心を行い上清を捨て沈澱をアスピレーターで１０分ほど乾燥させた。乾燥後、滅菌水３３．６μｌ、５Ｍ ＮａＣｌ ６．４μｌ、１３％ ＰＥＧ６０００ ４０μｌを加えてよく撹拌し氷上に２０分間静置した。これを１８，０００×ｇ、４℃、１５分間遠心し上清を注意深く捨て、沈澱に７０％エタノールを５００μｌ加えて洗浄しアスピレーターで乾燥させた。得られたＤＮＡに１０μｌの滅菌水を加えて溶解させ、Ａ_２６０、Ａ_２８０を測定してＤＮＡ濃度を計算し、−２０℃で保存した。
【００４４】
（５） ｃＤＮＡクローンの長さを調べるためのＰＣＲ
ｃＤＮＡクローンのｃＤＮＡ部分（インサート）の長さを調べるためにそれぞれのクローンをテンプレートとしてＰＣＲを行った。
【００４５】
実施例７ ＩＳＡ遺伝子の塩基配列の決定
テンプレートＤＮＡ（０．２５−０．５μｇ）に対して、Ｍ１３プライマーＦ、あるいはＲ（１ ｐｍｏｌ／μｌ）を３．２μｌ、Ｂｉｇ Ｄｙｅ ｐｒｅｍｉｘを４μｌ、希釈バッファー４μｌ、蒸留水を全体で２０μｌになるように加え、以下の条件でＰＣＲを行った。
ＰＣＲ後、ＰＣＲ産物に３Ｍ酢酸ナトリウムを２μｌ、９５％ エタノールを５０μｌ加え、室温で１５分間放置した。放置後、１８，０００×ｇ、２０分間遠心し、上清を残さないように注意深く吸い取り、沈殿に７０％エタノールを８００μｌ加え軽く撹拌した。１８，０００×ｇ、５分間遠心し上清を取り除き、アスピレーターで乾燥させた。ＨｉＤｉホルムアミドを２０μｌ加え、懸濁し、９５℃で２分間加熱した後氷冷し、シークエンサーにかけた。
得られた塩基配列を配列番号１に示す。得られた塩基配列から予測されるアミノ酸配列を配列番号２に示す。予想されるアミノ酸配列の一部を図１５に示す。
【００４６】
【発明の効果】
本発明は、新規なイソアミラーゼ、及びそれをコードする遺伝子を提供するものである。本発明のイソアミラーゼは紅藻類に由来するものであり、紅藻類はイネなどの高等植物が製造するデンプンとラン藻類が製造するフィトグリコーゲンとの中間的な構造を有するものであるから、本発明のイソアミラーゼと高等植物のデンプン合成酵素とを組み合わせることにより、新規なクラスター構造を有するデンプン類を製造できることになり、食糧用や産業用の新たな有用なデンプンを提供できる可能性がある。
【００４７】
【配列表】

【図面の簡単な説明】
【図１】図１は、ポルフィリディウム・プルプレウムＲ−１（Ｐｏｒｐｈｙｒｉｄｉｕｍ ｐｕｒｐｕｒｅｕｍ Ｒ−１）のデンプンの鎖長分布を示す。図１の横軸は鎖長（グルコースの数（ＤＰ））を示し、縦軸は各鎖長の量の全鎖長の合計の量に対する比率（％）を示す。
【図２】図２は、ガルディエリア・サルフラリアＭ−８（Ｇａｌｄｉｅｒｉａ ｓｕｌｐｈｕｒａｒｉａ Ｍ−８）のデンプンの鎖長分布を示す。図２の横軸は鎖長（グルコースの数（ＤＰ））を示し、縦軸は各鎖長の量の全鎖長の合計の量に対する比率（％）を示す。
【図３】図３は、シアニディウム・カルダリウムＮ−５５１（Ｃｙａｎｉｄｉｕｍ ｃａｌｄａｒｉｕｍ Ｎ−５５１）のデンプンの鎖長分布を示す。図３の横軸は鎖長（グルコースの数（ＤＰ））を示し、縦軸は各鎖長の量の全鎖長の合計の量に対する比率（％）を示す。
【図４】図４は、ゲリディウム（Ｇｅｌｉｄｉｕｍ（テングサ））のデンプンの鎖長分布を示す。図４の横軸は鎖長（グルコースの数（ＤＰ））を示し、縦軸は各鎖長の量の全鎖長の合計の量に対する比率（％）を示す。
【図５】図５は、イネ（Ｎｉｐｐｏｎｂａｒｅ）のデンプンの鎖長分布を示す。図５の横軸は鎖長（グルコースの数（ＤＰ））を示し、縦軸は各鎖長の量の全鎖長の合計の量に対する比率（％）を示す。
【図６】図６は、ポルフィリディウム・プルプレウム（Ｐ． ｐｕｒｐｕｒｅｕｍ）のゲル濾過クロマトグラフィの各画分のヨウ素デンプン反応による呈色反応の結果を示す図面に代わるカラー写真である。
【図７】図７は、紅藻ポルフィリディウム・プルプレウム（Ｐ． ｐｕｒｐｕｒｅｕｍ）のゲル濾過クロマトグラフィの結果を示す。図７の（ａ）（上段）はヨウ素デンプン反応における５９５ｎｍの吸光度による測定であり、（ｂ）（下段）はフェノール硫酸法による還元糖の定量による測定である。いずれもサンプルは２０ｍｇで、流速０．１４ｍＬ／分で、カラムはセファクリルＳ−１００（Ｓｅｐｈａｃｒｙｌ Ｓ−１００）を使用した。図７の（ａ）及び（ｂ）の横軸はフラクション番号を示し、（ａ）の縦軸は各フラクションの５９５ｎｍの吸光度を示し、（ｂ）の縦軸は各フラクションの４９０ｎｍの吸光度を示している。
【図８】図８は、紅藻ガルディエリア・サルフラリア（Ｇ．ｓｕｌｐｈｕｒａｒｉａ）のフェノール硫酸法による還元糖の定量による測定を示し、サンプルは２５ｍｇで、流速０．１６ｍＬ／分で、カラムはセファクリルＳ−１００（Ｓｅｐｈａｃｒｙｌ Ｓ−１００）を使用した。図８の横軸はフラクション番号を示し、縦軸は各フラクションの４９０ｎｍの吸光度を示している。
【図９】図９は、緑藻Ｃ． ｒｅｉｎｈａｒｄｔｉｉのゲル濾過クロマトグラフィの結果を示す。図９の、（ａ）（上段）はヨウ素デンプン反応における５９５ｎｍの吸光度による測定であり、（ｂ）（下段）はフェノール硫酸法による還元糖の定量による測定である。いずれもサンプルは２０ｍｇで、流速０．１４ｍＬ／分で、カラムはセファクリルＳ−１００（Ｓｅｐｈａｃｒｙｌ Ｓ−１００）を使用した。図９の、（ａ）及び（ｂ）の横軸はフラクション番号を示し、（ａ）の縦軸は各フラクションの５９５ｎｍの吸光度を示し、（ｂ）の縦軸は各フラクションの４９０ｎｍの吸光度を示している。
【図１０】図１０は、緑藻Ｃ． ｋｅｓｓｌｅｒｉのゲル濾過クロマトグラフィの結果を示す。図１０の、（ａ）（上段）はヨウ素デンプン反応における５９５ｎｍの吸光度による測定であり、（ｂ）（下段）はフェノール硫酸法による還元糖の定量による測定である。いずれもサンプルは２０ｍｇで、流速０．１４ｍＬ／分で、カラムはセファクリルＳ−１００（Ｓｅｐｈａｃｒｙｌ Ｓ−１００）を使用した。図１０の、（ａ）及び（ｂ）の横軸はフラクション番号を示し、（ａ）の縦軸は各フラクションの５９５ｎｍの吸光度を示し、（ｂ）の縦軸は各フラクションの４９０ｎｍの吸光度を示している。
【図１１】図１１は、イネ（Ｆｕｊｉｈｉｋａｒｉ）のゲル濾過クロマトグラフィの結果を示す。図１１の、（ａ）（上段）はヨウ素デンプン反応における５９５ｎｍの吸光度による測定であり、（ｂ）（下段）はフェノール硫酸法による還元糖の定量による測定である。いずれもサンプルは２０ｍｇで、流速０．１４ｍＬ／分で、カラムはセファクリルＳ−１００（Ｓｅｐｈａｃｒｙｌ Ｓ−１００）を使用した。図１１の各々の、（ａ）及び（ｂ）の横軸はフラクション番号を示し、（ａ）の縦軸は各フラクションの５９５ｎｍの吸光度を示し、（ｂ）の縦軸は各フラクションの４９０ｎｍの吸光度を示している。
【図１２】図１２は、ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）とイネ（Ｆｕｊｉｈｉｋａｒｉ）のゲル濾過クロマトグラフィで得られたフラクションのアミロペクチンとアミロースの画分の鎖長分布を解析した結果を示す。図１２の（ａ）（上段）はアミロペクチン画分の比較であり、（ｂ）（下段）はアミロース画分の比較である。図１２の灰色の棒はイネ（Ｆｕｊｉｈｉｋａｒｉ）を示し、黒色の棒は紅藻ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）を示す。図１２（ａ）及び（ｂ）の横軸は鎖長（グルコースの数（ＤＰ））を示し、縦軸は各鎖長の量の全鎖長の合計の量に対する比率（％）を示す。
【図１３】図１３は、ヨウ素デンプン反応を利用した活性染色（Ｎａｔｉｖｅ ＰＡＧＥ）により、ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）のＩＳＡの活性を試験した結果を示す図面に代わる写真である。図１３レーン１及び２は、マーカーとしてのイネの登熟種子抽出液であり、レーン３は紅藻ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）の細胞をガラスビーズで破砕した破砕液であり、レーン４はその１８，０００×ｇでの遠心分離の上澄液である。図１３の左側にはプルラナーゼ（ＰＵＬ）の位置を示し、右側にはイソアミラーゼ（ＩＳＡ）の位置を示している。
【図１４】図１４は、ＩＳＡ遺伝子に共通に見られる領域（保存領域）を示す。図１４中に例示しているのはトウモロコシ（ｍａｉｚｅ ｅｎｄｏｓｐｅｒｍ）、イネ（ｒｉｃｅ ｅｎｄｏｓｐｅｒｍ）、ラン藻（Ｓｙｎｅｃｈｏｃｙｓｔｉｓ）の２種である。図１４の下段は、本発明の紅藻のＩＳＡのためのＰＣＲのプライマーを示している。
【図１５】図１５は、本発明のイソアミラーゼ（ＩＳＡ）の予想されるアミノ酸配列の一部を、アミノ酸の１文字表記により示したものである。
【図１６】図１６は、各種の生物のＩＳＡと本発明のＩＳＡのアミノ酸配列の比較を示す。図１６の上から本発明の紅藻ポルフィリディウム・プルプレウム（Ｐ．ｐｕｒｐｕｒｅｕｍ）の解読できた部分のアミノ酸配列を示し、その下の段はイネのＩＳＡ（Ｒｉｃｅ−ＩＳＡ）を示し、その下の段はラン藻ｇｌｇＸのＩＳＡ（Ｓｃｙｓｔｉｓ−ｇｌｇＸ）を示し、その下の段はラン藻ｇｌｇＸＸのＩＳＡ（Ｓｃｙｓｔｉｓ−ｇｌｇＸＸ）を示し、その下の段はＰｓｅｕｄｏｍｏｎａｓ ａｍｙｌｏｄｅｒａｍｏｓａのＩＳＡ（Ｐ．ａｍｙｌｏ−ＩＳＡ）を示す。
【図１７】図１７、各種の生物のＩＳＡと本発明のＩＳＡのアミノ酸配列の比較を示す図１６の続きである。
【図１８】図１８は、本発明の方法で得られたクローン１からのＩＳＡの配列に他の生物のＩＳＡの配列を加えて作製した系統樹を示す。図１８の各枝の上の数字はブーツストラップを示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an isoamylase derived from Porphyridium, a kind of red algae, a gene encoding the same, and a vector and a transformant containing the gene. The present invention also relates to an oligonucleotide comprising a partial sequence of a base sequence of a gene encoding isoamylase derived from Porphyridium, a kind of algae.
The isoamylase of the present invention is one kind of an enzyme that synthesizes a starch specific to red algae, and it is possible to develop a food or industrial starch having a novel structure by utilizing the enzyme activity. It becomes possible.
[0002]
[Prior art]
Starch is used not only as a main ingredient of grains but as food and feed, but also processed into dextrins, oligosaccharides, isomerized sugars, etc., and used in processed foods. It is also used as a raw material. Even if it is simply referred to as starch, rice starch, potato starch, wheat starch, corn starch, etc., the form, taste, physical properties when gelatinized, etc. of the starch differ slightly depending on its origin. Various plant-derived starches have been used for different purposes. It has been explained that such differences in the properties of starch stem from differences in the chemical structure of starch.However, starch is generally made of amylose and amylopectin. It is said to be largely derived from amylopectin having
Starch (Starch) is an α-polyglucan that accumulates in storage organs of higher plants. Starch is composed of two types of glucose homopolymers, amylopectin and amylose. Amylose is a linear helical molecule in which glucose units are linked by α-1,4 glucosidic bonds and contain small amounts of α-1,6 glucosidic bond branches. On the other hand, amylopectin has a structure in which a glucose unit is extended by α-1,4 glucosidic bonds, and a branch is extended from the main chain by α-1,6 glucosidic bonds. These branches form a cluster to form a characteristic structure called a “cluster” structure.
Glycogen, which is a storage substance of animals and bacteria, is also composed of glucose homopolymer like amylopectin, but does not have a cluster structure and has a random structure called "tree like" or "bush like". have. Glycogen is composed of a completely irregular branched structure. Glycogen has smaller molecules and shorter branches than amylopectin, and many of them are water-soluble substances. Amylopectin, on the other hand, has long branches and is filled with glucose at a high density, and is generally a water-insoluble substance.
[0003]
The outline of the synthesis of starch is as follows: (1) Starch synthase (SS), (2) Starch branching enzyme (SBE), (3) Starch branching enzyme (Starch). It is synthesized by the reaction of debranching enzyme (DBE). SS connects ADP glucose to the non-reducing end of amylopectin via an α-1,4 glucoside bond, and plays a role in extending the chain. While SS extends the chain of amylopectin, SBE is an enzyme that forms an α-1,6 glucoside bond of amylopectin, and is an enzyme that forms a branched portion of a branched structure. Heretofore, starch debranching enzyme (DBE) was considered to be an enzyme that was not required for cluster formation. However, it has been clarified that a plant lacking this degrading enzyme cannot form amylopectin cluster, and it has been reported that DBE is essential for the formation of a cluster structure (Non-Patent Documents 1 to 3). 3).
[0004]
As described above, the biosynthesis of amylopectin, which is a component of starch, requires the presence of isoamylase, which is a type of DBE. Therefore, higher plants that synthesize starch have isoamylase corresponding to each plant. . Organisms that do not produce starch, such as animals, do not have isoamylase, but some organisms, which do not produce starch, do. The reason for the existence of isoamylase in such organisms is not clear at present, and further research is expected. To date, the isoamylase (ISA) gene of cyanobacteria has been isolated (see Patent Document 1), but no isoamylase has been isolated from other primitive organisms.
[0005]
Red algae is a photosynthetic organism created by the coexistence of cyanobacteria with eukaryotic cells that are the ancestors of red algae, and is said to be one of the oldest eukaryotes. The chloroplasts of red algae are derived from green plants, but have no genetic affinity with green plants. This is thought to be the result of the host, which is not genetically related to the green plant, taking in the green plant and symbiotic. Red algae are an organism of great interest both in the evolution of organisms and in the taxonomics of organisms.
Red algae are marine and freshwater, and are known to be extremely diverse from microalgae to macroalgae. Red algae are taxonomically belonging to the Rhodophyceae of the Rhodophyta of Eukaryophyta, of which about 600 genera and 5000 species are currently known. About 200 species are known as freshwater ones.
[0006]
However, there is little information on the starch synthesis and metabolic system of red algae, and studies on the metabolism of carbohydrates of red algae have been conducted since the 1960s. At that time, research was mainly conducted on red alga starch of the macroalga Dilsea edulis, and Fredrick et al. (1971) were conducting research on isozymes of glucose transferase (Glucocyltransferase) of Cyanidium caldarium, a primitive red algae (see Non-Patent Document 4). At this time, the red algae starch has a red algae starch (fluoridean Starch) unique to red algae due to the coloration, viscosity, and average chain length of the iodine starch reaction, and has no amylose structurally, only amylopectin. It was said to be a constituent. However, in the 1980's, it was suggested that amylopectin and amylose were present in the starch of the red alga Glaucosphaera vacuolata (see Non-Patent Document 5). Since then, red algae starch has been divided into two types: protofluorideoacidae starch, which is thought to have amylose in the starch granules, and florideophycide starch, including red alga starch. It became so. However, the primordial red algae Cyanidiosizon merole and C. It has been reported that caldarium has a phytoglycogen-like starch structure, and the starch structure of red algae has not been clearly elucidated. The mechanism of starch synthesis has been elucidated, and the development of a starch having a novel amylopectin structure has been reported. Therefore, elucidation of the starch structure of red algae and elucidation of its enzyme system are required.
[0007]
[Patent Document 1]
JP-A-2002-262877
[Non-patent document 1]
M. G. FIG. James, et al. , Plant Physiol. , (1995) 7, 417-429.
[Non-patent document 2]
Y. Nakamura, et al. , Plant J .; , (1997) 12 (1), 143-153.
[Non-Patent Document 3]
A. Kubo, et al. , Plant Phys. , (1999) 121, 399-409.
[Non-patent document 4]
Fredrick, J .; F. , Phytochemistry, (1971) 10, 395-398.
[Non-Patent Document 5]
McCracken, D.C. A. , Et al. , New Phytol. , (1981) 88, 67-71.
[0008]
[Problems to be solved by the invention]
An object of the present invention is to analyze the structure of a red algae starch and to isolate and identify its synthase. Red algae starch has a unique structure that is different from other organisms.Not only is it itself useful as a novel starch for food and industrial use, but its synthase is a starch with a novel structure. It is useful as an enzyme for producing.
[0009]
[Means for Solving the Problems]
The present inventors have succeeded in isolating a novel isoamylase gene possessed by red algae from structural analysis of red algae starch, identified isoamylase, and reached the present invention.
[0010]
TECHNICAL FIELD The present invention relates to an isoamylase derived from Porphyridium, a kind of red algae. The present invention also relates to a gene encoding the isoamylase, a vector containing the same, and a transformant containing the same.
Furthermore, the present invention relates to a partial sequence of the nucleotide sequence of the gene, more specifically, an oligonucleotide comprising at least 10 consecutive nucleotides or more.
[0011]
The present inventors have proposed, as biological materials, Porphyridium purpureum R-1 and Gardieria sulphuraria M-8 belonging to the primordial red algae subclass (Protoflorideophycidae). Calyanium N-551 (Cyanidium caldarium N-551), and porphyra (Porphyra (Susabinori)), and a geridium (Glycium spp.) Belonging to the subfamily Florideophycidae Then, the structure of the starch was analyzed, and the porphyridium purpurium R-1 (Porphyridium purpurium) therein was analyzed. A new isoamylase was successfully isolated from R-1).
[0012]
The present inventors cultured these organisms for several days, centrifuged the cells, crushed the cells, and centrifuged the obtained cell lysate to isolate starch from each cell.
The chain length of each starch of these red algae was analyzed and compared with the starch chain length distribution of the higher plant rice (Nipponbare).
Porphyridium purpurium R-1; Gardieria sulphularia M-8; Cyanidium cardarium N-551 (Cyanidium calidarium N-551 and Cyanidium calidarium N-551). FIGS. 1 to 5 show the results of starch chain length analysis performed on Tengsa) and rice (Nipponbare), respectively. The horizontal axis in the chain length distribution diagrams in FIGS. 1 to 5 indicates the chain length (number of glucose (DP)), and the vertical axis indicates the ratio (%) of the amount of each chain length to the total amount of the total chain length. Show. FIG. 1 shows the starch of Porphyridium purpureum R-1; FIG. 2 shows the starch of Gardieria sulphularia M-8; FIG. 3 shows the starch of Porphyridium sulphuraria M-8; FIG. 4 shows starch of Calidium N-551, FIG. 4 shows starch of Gelidium (Tengsa), and FIG. 5 shows starch of rice (Nipponbare).
[0013]
As a result, despite the same primordial red algae subclass, there is a chain between G. sulphuraria, C. caldarium and P. purpureum. Differences were found in the length distribution. Among them, G. sulfuraria and C. caldarium were thought to have many branches with a short chain length of DP 10 or less and to be close to the chain length distribution of cyanobacteria. However, a prominent peak is seen near DP8, and there is a possibility that impurities such as oligosaccharides are mixed. On the other hand, the chain length distribution of Porphyridium purpureum (P. purpureum) is similar to that of Gelidium of the genus Rhodophyta, both of which have more short chains than rice and have a lower DP10-15 chain length peak, and have a cluster structure. The peak of DP45, which is considered to be necessary for forming, was lower than that of rice.
[0014]
Next, the red algae P. purpurium, G. sulphuraria, the green alga Chlamydomonas reinhardtii CC125, the Chlorella keseliri 11h, and the higher plant rice filtration (Fujihden) were chromatographed on Kujiri Denki. Was. Red algae Porphyridium purpureum (P. purpureum), green alga C. reinhardtii, C.I. For Kessleri, starch obtained by the Percoll method was used, and for the red algae G. sulphuraria, starch obtained by the DMSO extraction method was not obtained, because starch was not obtained by the Percoll method. Was used.
The results of the color reaction of each fraction of the red alga Porphyridium purpurium (P. purpurum) by the iodine starch reaction are shown in FIG. Fractions 11-13 exhibited purple coloration, and fractions 19-25 exhibited blue coloration. FIG. 7 shows the results of gel filtration chromatography of the red alga Porphyridium purpurium. FIG. 7 (a) (upper) shows the measurement based on the absorbance at 595 nm in the iodine starch reaction, and FIG. 7 (b) (lower) shows the measurement based on the quantitative determination of reducing sugars by the phenol-sulfuric acid method. In each case, the sample was 20 mg, the flow rate was 0.14 mL / min, and Sephacryl S-100 was used as the column. The horizontal axis of (a) and (b) of FIG. 7 shows the fraction number, the vertical axis of (a) shows the absorbance of each fraction at 595 nm, and the vertical axis of (b) shows the absorbance of each fraction at 490 nm. ing.
As can be seen from the figure, two peaks were obtained from the measurement of the absorbance at 595 nm, and the λmax in each peak fraction (fraction 12 and fraction 22) was 540 nm and 639 nm (see FIG. 7A). Compared to λmax of higher plants, the peak at 540 nm is considered to be amylopectin, and the peak at 639 nm is considered to be amylose. Further, the absorbance at 490 nm due to coloration by the phenol-sulfuric acid method was measured (see FIG. 7B). The graph of 595 nm and the graph of the phenol sulfate method are almost the same, considering that amylose is more likely to be stained about 20 times than amylopectin. A comparison of the amount of reducing sugars between amylopectin and amylose based on the results of the phenol-sulfuric acid method revealed that amylopectin was considerably higher.
[0015]
In the red alga Gardieria Sulfuraria (G. sulphuraria), no coloration due to the iodine starch reaction was observed. This is presumably because the starch in G. sulphuraria has a structure that is difficult to stain with iodine, or that the starch is not in an amount sufficient for staining with iodine. Since no coloration due to the iodine starch reaction was observed, it was thought that the amount of reducing sugar was small. In the phenol-sulfuric acid method, different from the usual conditions, each fraction was reacted at 50 μl and 0.5 M HCl at 10 μl. . As a result, a single peak was obtained. FIG. 8 shows the results. Fig. 8 shows the measurement of the reducing algae of the red alga Gardieria Sulfuraria by the phenol-sulfuric acid method. The sample was 25 mg, the flow rate was 0.16 mL / min, and the column was Sephacryl S-100 (Sephacryl S). -100) was used. The horizontal axis in FIG. 8 shows the fraction number, and the vertical axis shows the absorbance at 490 nm of each fraction.
This suggests that, unlike the porphyridium purpurum (A. purpureum) starch containing amylopectin and amylose, the G. suphuraria starch is composed of a single component.
[0016]
Similarly, green alga C. reinhardtii and C.I. Gel filtration chromatography was performed on kessleri starch and rice starch.
FIG. Fig. 9 shows the results of gel filtration chromatography of reinhardtii. FIG. 2 shows the results of gel filtration chromatography of Kessleri. FIG. 11 shows the results of gel filtration chromatography of rice (Fujihikari).
In each of FIGS. 9 to 11, (a) (upper row) is a measurement based on absorbance at 595 nm in the iodine starch reaction, and (b) (lower row) is a measurement based on quantification of reducing sugars by the phenol-sulfuric acid method. In each case, the sample was 20 mg, the flow rate was 0.14 mL / min, and Sephacryl S-100 was used as the column. 9 to 11, the horizontal axis of (a) and (b) indicates the fraction number, the vertical axis of (a) indicates the absorbance of each fraction at 595 nm, and the vertical axis of (b) indicates the fraction of each fraction. The absorbance at 490 nm is shown.
[0017]
Green algae C.I. reinhardtii and C.I. Kessleri starch had amylopectin and amylose similarly to rice starch (see FIGS. 9 (a) and 10 (a)). The results of the phenol-sulfuric acid method of these green algae were slightly higher than those of the red alga P. purpureum (P. purpureum), but the green algae had a higher amylose content (C. reinhardtii 22.4%, C. 13.0%, Porphyridium purpurium (6.4%) (see FIGS. 9 (b) and 10 (b)). In the case of rice, both the iodine starch reaction and the phenol-sulfuric acid method showed higher amylose peaks and higher contents (amylose content 29.4%) than red algae and green algae (FIGS. 11 (a) and (b)). reference).
Table 1 shows the results obtained by measuring λmax near the peak obtained from the iodine starch reaction, averaging the results, and generating a standard error.
[0018]
[Table 1]

[0019]
Next, the chain length distribution of the fractions of amylopectin and amylose in the fractions obtained by gel filtration chromatography of porphyridium purpureum (P. purpureum) and rice (Fujihikari) was analyzed. FIG. 12 shows the chain length distribution of both. (A) (upper) of FIG. 12 is a comparison of the amylopectin fraction, and (b) (lower) is a comparison of the amylose fraction. The gray bars in FIG. 12 indicate rice (Fujihikari), and the black bars indicate the red alga Porphyridium purpurium. 12A and 12B, the horizontal axis indicates the chain length (number of glucose (DP)), and the vertical axis indicates the ratio (%) of the amount of each chain length to the total amount of the total chain length.
In the case of P. purpureum, although the amount of the short difference was slightly larger in the amylose fraction than in the amylopectin fraction, both fractions showed very similar chain length distributions. This is probably because the chain length distribution of amylose alone could not be obtained due to the mixture of amylopectin in the amylose fraction. In this regard, the same was said for Fujihikari.
In addition, amylopectin of P. purpureum (P. purpureum) had more short chains of DP8 or less than that of Fujijikari, but had less distribution around DP10-25. In addition, a difference was also observed in the vicinity of DP45, where a gentle peak was observed in Fujihikari, whereas no peak was observed in porphyridium purpurium.
[0020]
From the above, the starch of the red alga Porphyridium purpureum (P. purpureum) has a larger number of short chains than that of rice, and has a long chain near DP45 which is considered necessary for forming a cluster structure. It can be seen that the peak is low and that of long chains is small. It is known that cyanobacteria mainly have a very large number of short chains with a DP of 10 or less, and do not have a peak near DP45, which is considered to be necessary for forming a cluster structure. Although there was a slight difference, the chain length distribution was similar to that of higher plant rice, and a peak near DP45 was also observed, indicating that a cluster structure was formed. There is a slight difference in the short chain between rice and green algae, but the long chain peak near DP45 is commonly observed, so that green algae form a starch structure closer to higher plants than red algae. It is thought that it is. Since cyanobacteria synthesize phytoglycogen, in chain length analysis, there are many short chains up to around DP3-8, and many have no long chains after DP30.
This means that Porphyridium purpureum (P. purpureum) has fewer short chains compared to cyanobacteria, but has long chains after DP30. From these facts, it is considered that the starch of P. purpureum is an intermediate type between cyanobacterium and rice. Structurally, the structure is not as firm as the cluster structure of rice amylopectin, but it is thought that it does not branch off as randomly as the phytoglycogen of cyanobacteria. On the other hand, the red alga C. caldarium and G. sulphuraria, which have a different chain length distribution from that of P. purpureum, have very short chains, and have a very short chain length. It is considered that the chain length distribution is close to that of algae.
Gelidium, a large algae, has few side chains around DP10-15, but the rest is very similar to the chain length analysis in the case of P. purpureum. Is thought to contain a structure more similar to that of P. purpurumum than that of C. caldarium or G. sulfuraria.
[0021]
Thus, the existence of some red algae with or without amylose has been clarified. In particular, in the case of Gardieria sulphuraria (G. sulphuraria), no coloration was obtained in the iodine starch reaction despite the presence of a reducing sugar confirmed by the phenol-sulfuric acid method. In addition, comparing the elution positions of porphyridium purpurium (P. purpureum) starch by gel filtration chromatography, porphyridium purpureum (P. purpureum) elutes from around 40 ml, whereas Gardieria In G. sulphuraria, it was eluted from around 80 ml. From these facts, the polyglycan of G. sulphuraria is composed of one constituent, is hardly stained by the iodine starch reaction, and is smaller than the amylopectin fraction of P. purpureum. It can be seen that it has a molecular weight. The construct is considered to have an incomplete amylopectin structure or a phytoglycogen-like structure, and it is considered to have a phytoglycogen-like structure when the results of chain length analysis are added.
It was also found that both red algae and green algae had significantly less amylose than rice.
[0022]
We believe that isoamylase (ISA) is essential for the formation of starch cluster structures in higher plants. Therefore, the existence of ISA was examined in the red alga Porphyridium purpurium, in which the presence of a starch cluster structure was confirmed.
FIG. 13 shows a photograph as a substitute for a drawing, in which the ISA activity of P. purpureum was tested by activity staining (Native PAGE) using an iodine starch reaction. FIG. 13 Lanes 1 and 2 are ripening seed extracts of rice as a marker, and Lane 3 is a crushed liquid obtained by crushing cells of a red alga Porphyridium purpureum (P. purpureum) with glass beads. 4 is the supernatant of centrifugation at 18,000 × g. The left side of FIG. 13 shows the position of pullulanase (PUL), and the right side shows the position of isoamylase (ISA).
As a result, it was possible to confirm the presence of ISA in the red alga Porphyridium purpurium (see lanes 3 and 4 in FIG. 13). This suggests that ISA is involved in the synthesis or degradation of amylopectin even in P. purpureum as in green plants.
In this activity staining, amylopectin in the gel is apt to be stained with iodine when the α-1,6 glycosidic bond is cleaved by ISA and the number of branches is reduced, and the amylopectin shows a blue color. It is degraded into sugars such as maltose and oligosaccharides, and is not stained by iodine. Therefore, in FIG. 13, several bands appearing white in color are observed, which are considered to be amylase.
[0023]
It is considered that red algae has a unique starch metabolism system, such as the fact that UDP-glucose is used as a glucose donor, and there are those that synthesize amylose and those that do not. Therefore, among the starch synthesis-related enzymes, the cDNA of ISA, which could be confirmed by the above experiment, was first cloned and its nucleotide sequence was determined, and compared with cyanobacteria, green algae and higher plants, I decided to consider if there were any differences.
[0024]
It is known that ISA has a region (conserved region) commonly found in ISA genes. This is shown in FIG. Illustrated here are two types of maize (maize endosperm), rice (rice endosperm), and cyanobacteria (Synechocytis). In each case, there are conserved areas in the four areas I, II, III, and IV shown in FIG.
Thus, PCR was performed using primers prepared based on the amino acid sequence of a region (conserved region) commonly found in the ISA gene, and using the cDNA of the red alga Porphyridium purpurium as a template. . When PCR was performed using various combinations of primers, PCR was performed using the F1 and R1 primers, and then nested PCR was performed using the PCR product as a template and F1 and R2 primers (shown in the lower part of FIG. 14). The PCR product of the expected size was obtained. The obtained PCR fragment was sequenced. Since the amino acid sequence predicted from the obtained nucleotide sequence showed homology to that of ISA of other organisms, a 68-mer probe was prepared based on the nucleotide sequence (sequence between Box I and II) of this portion. .
[0025]
About 5 × 10 ⁵ When the clones were screened, five positive clones were obtained in the primary screening, and positive clones were obtained in all five types in the secondary screening. Of the five positive clones, two had successful in vivo excision, and four had different dilutions during in vivo excision. Was. Two were taken from the plate and a total of eight were sequenced. As a result of the sequencing, three clones having different sequences were obtained, except for the clones having the same sequence among the eight clones.
Of the three clones obtained, clone 2 was highly homologous to Arabidopsis thaliana's DegP protein (AY039598-1) from homology search, and thus analysis was terminated.
Clone 1 and clone 3 contained a sequence that matched the sequence of the PCR product described above.
The nucleotide sequence deciphered from these clones is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence predicted to encode the gene is shown in SEQ ID NO: 2. FIG. 15 shows the amino acid sequence in one letter code of amino acids.
[0026]
The homology between the nucleotide sequences of clone 1 and clone 3 was 99.4%, and the amino acid sequences predicted from the nucleotide sequences of these clones showed high homology with those of ISAs of other organisms. FIGS. 16 and 17 show a comparison of the amino acid sequences of ISAs of various organisms and the ISA of the present invention. FIG. 16 and FIG. 17 show the amino acid sequence of the portion of the red alga Porphyridium purpurium of the present invention that could be decoded from above, and the lower row shows the rice ISA (Rice-ISA). , The lower row shows the ISA of cyanobacterial glgX (Scysis-glgX), the lower row shows the ISA of cyanobacterial glgXX (Scysis-glgXX), and the lower row shows the ISA of Pseudomonas amyloderamosa (P. amylomo). -ISA). FIG. 17 is a continuation of FIG. 16.
FIG. 18 shows a phylogenetic tree prepared by adding the sequence of clone 1 to the sequence of ISA of another organism. The numbers above each branch in FIG. 18 indicate a boot strap.
[0027]
The present invention provides an isoamylase derived from the genus Porphyridium of the primordial red alga Subphylum, Porphyridiales, more specifically, from a species of Purpyridium purpureum, and a gene encoding the same. Things. The present invention confirms the presence of isoamylase of red algae and clones a gene encoding the enzyme, and the entire nucleotide sequence has not been completely analyzed, but by the method described herein. The gene of the target red algae can be obtained, and its base sequence can be decoded by a conventional method.
In the above example, the isoamylase of the present invention is derived from R-1 of P. purpurumum, but is not limited thereto.
As described above, the isoamylase of the present invention can form a structure as tight as the cluster structure of rice amylopectin, such as the intermediate type between cyanobacteria and rice, which is a starch unique to red algae. Although not yet, it is considered that starch having a structure not branched as randomly as phytoglycogen of cyanobacteria is useful for producing starch. In general, starch with a loose cluster structure is more water-soluble than starch with a firm structure, and has specific properties such as gelatinization temperature, which is important for the production of completely new starch. It is considered an enzyme.
Although the isoamylase of the present invention has a partial amino acid sequence (see SEQ ID NO: 2), it is not limited to those having this amino acid sequence, and it is not limited to those having a red algae, preferably of the order of Porphyridiales. More preferably, it is derived from the genus Porphyridium, even more preferably from a species of Purpyridium purpureum, and may be any as long as it has an activity capable of forming starch specific to the aforementioned red algae.
[0028]
The isoamylase of the present invention can be produced by the method described above, but is not limited to this method, and may be a combination of a known method such as a PCR method using the base sequence provided by the present invention. Can be produced by various methods.
Further, the method for cloning the gene of the present invention is not limited to the above-described method, and various known cloning methods can be used.
Furthermore, the isoamylase gene of the present invention can be used by connecting genes having various base sequences upstream and / or downstream thereof, if necessary. In any case, the gene of the present invention can be used. Is included in the technical scope of the present invention as long as it contains. Examples of genes that can be connected to the upstream side of the isoamylase gene of the present invention include promoters derived from various organisms. The promoter of the red alga Porphyridium purpurumum (P. purpureum) can also be used as the promoter, but those of other organisms can also be used in combination.
[0029]
The present invention includes a vector containing the isoamylase gene of the present invention described above. The vector may be a cloning vector as described in the above example, or may be an expression vector to which a promoter is linked. Various plasmids can be selected as a target.
The present invention also provides a transformant into which the above-described gene of the present invention has been introduced. The host cell into which the gene of the present invention is introduced is not particularly limited, but a cell capable of producing starch is preferable. When the host cell already has the isoamylase gene, it may be knocked out or the gene of the present invention may be introduced as it is. Cells into which the gene of the present invention has been introduced and expressed in this way are subjected to starch debranching by the activity of the isoamylase of the present invention, thereby producing starch having a structure similar to that of red algae starch. Conceivable.
[0030]
The present invention also provides an oligonucleotide comprising a partial sequence of the isoamylase gene of the present invention. The oligonucleotide of the present invention is useful as a primer in PCR, a cloning probe, a probe for detecting the presence of the gene of the present invention, or the like. Although the length of the oligonucleotide of the present invention is not particularly limited, preferably those having a length of 10 to 150 bases, 15 to 150 bases, 20 to 150 bases, 15 to 50 bases or the like can be appropriately used.
[0031]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Biological materials porphyridium purpurium R-1 (tinorimo), Gardieria sulphuraria M-8, and cyanidium cardarium N-551 (Cyanidium diumdium). 551) were obtained from the IAM Culture Collection of the Institute of Molecular and Cellular Sciences, the University of Tokyo. Sushibinori (Porphyra sp.) Was obtained from the Utsumi Castle Functional Education and Research Center, Kobe University. Tengsa (Gelidium sp.) Was collected from the beach of Ehime.
Porphyridium purpureum (P. purpureum) in MKM medium at 20 ° C., 2% CO 2 ₂ , Culture of G. sulfuraria in Allen medium at 33 ° C. and air bubbling, and cyanidium cardarium in C. caldarium at A15L medium at 33 ° C. and air bubbling. did.
[0032]
Example 1 (starch extraction)
(1) Extraction from small organisms
Cells such as Porphyridium purpurium cultured for several days were centrifuged at 1,700 × g at 4 ° C. for 10 minutes. The cells were crushed by adding methanol and vortexing with glass beads for 10 minutes. After centrifugation at 1,700 × g at 4 ° C. for 10 minutes, methanol was again added to the precipitate, and the above operation was repeated until the dye was removed. Dimethyl sulfoxide (DMSO) was added to the centrifuged precipitate, and the mixture was boiled at 90 ° C. for 120 minutes. After boiling, the mixture was transferred to a 50 ml tube, centrifuged at 1,700 × g at 4 ° C. for 10 minutes, the supernatant was placed in a 1 L medium bottle, DMSO was added again to the precipitate, and the mixture was boiled at 90 ° C. for 120 minutes. After boiling, the above-mentioned centrifugation operation was performed once again. The obtained supernatant was placed in a 1 L medium bottle, and about 800 ml of ethanol was added thereto. The mixture was left overnight at -20 ° C to precipitate starch.
[0033]
(2) Extraction from macroalgae
Large algae such as Porphyra sp were placed in a mortar, frozen with liquid nitrogen and ground with a pestle. After grinding to some extent, methanol was added and the mixture was stirred and kept at -20 ° C overnight. The next day, the mixture was centrifuged at 2,000 × g for 10 minutes, and the precipitate was placed in a mortar and the same operation as the previous day was performed to disrupt the cells as much as possible. The cell lysate was centrifuged at 2,000 × g for 10 minutes to remove methanol, and DMSO was added. The subsequent operation was performed in the same manner as in the extraction of starch from microalgae.
[0034]
Example 2 (Analysis by capillary electrophoresis)
Aggregates of α-1,4 glucans of various lengths obtained by completely decomposing the α-1,6 glycosidic bond of starch with isoamylase were obtained. ATPS (1-aminopyrene-3,6,8-trisulfonic acid trisodium salt) as a fluorescent substance was added to the reducing terminals of all the α-1,4 glucans obtained by the enzyme treatment, and they were electrophoresed to 488 nm. Quantitative detection with high sensitivity using a laser-induced fluorescence detector.
The results are shown in FIGS. As a comparison, FIG. 5 shows the chain length distribution of rice starch.
[0035]
Example 3 (Structure analysis of starch by gel filtration chromatography)
(1) Sample preparation
The microalga starch was suspended by adding 1 ml of ethanol, centrifuging at 10,000 × g, 4 ° C. for 10 minutes, removing the supernatant, and drying with an aspirator.
Rice (Fujihikari) starch was obtained by placing endosperm in a mortar, grinding with a pestle, adding ethanol, suspending the suspension, and centrifuging at 18,000 × g for 5 minutes. The precipitate was dried with an aspirator to obtain starch.
To 20 mg of starch, 1.6 ml of distilled water was added to suspend, and 0.4 ml of 5N NaOH was added and mixed. The starch was left in a 47 ° C. water bath for 30 minutes to gelatinize the starch. 2.0 ml of distilled water was added and mixed, centrifuged at 2,000 × g for 5 minutes at room temperature, and 3.0 ml of the supernatant was transferred to another test tube to obtain a gel filtration sample.
(2) Gel filtration chromatography
Structural analysis by gel filtration chromatography was performed according to the method of Nakamura et al. (Nakamura, Y., et al., Plant J., (1997), 12 (1), 143-153). 60 preparative test tubes (6 ml) were set in a fraction collector (ATTO). The flow rate was about 0.14 ml / min, and about 4 ml was fractionated per test tube.
FIG. 6 shows the results of a color reaction by iodine starch reaction of each fraction of the obtained porphyridium purpurium (P. purpureum) by gel filtration chromatography.
7 to 11 show the results of the respective gel filtration chromatography.
[0036]
Example 4 (Chain length analysis of each fraction in gel filtration)
Among the fractions obtained by gel filtration chromatography, fractions estimated to be amylopectin and amylose from the measurement of λmax were respectively collected in medium bottles, ethanol was added thereto, and the mixture was allowed to stand overnight at -20 ° C to precipitate starch. The starch was recovered by centrifugation, and the chain length was analyzed by capillary electrophoresis as described above.
The results are shown in FIG.
[0037]
Example 5 (Activity staining of starch synthesis / degradation-related enzymes (Native PAGE))
In order to examine the activity of enzymes involved in starch synthesis / degradation, electrophoresis was performed using a native gel, and activity staining was performed.
An acrylamide gel was prepared as follows.
Potato amylopectin (24 mg) was weighed into a beaker, 3.3 ml of distilled water was added, mixed, covered with plastic wrap, and dissolved with stirring with a stirrer at 60 ° C. until clear. 1.65 ml each of 30% acrylamide, 0.8% bis-acrylamide, 1.5 M Tris-HCl (pH 8.8), 0.2% TEMED and 3.3 ml of amylopectin solution were mixed, and 10% AMPS (ammonium persulfate) was mixed. ) Was added and poured into a gel plate. Butanol was overlaid and left for about 30 minutes. After 30 minutes, butanol was removed, and the upper part of the gel was washed with distilled water. 0.3% each of 30% acrylamide, 0.8% bis-acrylamide, 1.0 M Tris-HCl (pH 6.8), 0.2% TEMED, 1.8 ml of distilled water, mix, add 16 μl of APS and gel The mixture was poured into a plate, a comb of 10 or 12 lanes was inserted, and left for 20 minutes.
The rice (ripening) paddy and embryos were removed, placed in an Eppendorf tube, and the endosperm weight was measured. In the endosperm, a buffer for cell disruption (Grind Solution (hereinafter abbreviated as Gs)) (50 mM imidazole-HCl (pH 7.4), 8 mM MgCl ₂ , 12.5% glycerol, 500 mM 2-mercaptoethanol) and ground with a plastic pestle. The mixture was centrifuged at 18,000 × g at 4 ° C. for 20 minutes, and the supernatant was placed in another tube. This supernatant was mixed with the sample buffer at a ratio of 2: 1 to obtain a migration marker.
The cells of the sample were collected at 1,700 × g for 10 minutes. The collected cells were suspended in a cell disruption buffer (Gs). The suspended cell solution was placed in a test tube containing glass beads, vortexed for 10 minutes to break the cells, and centrifuged at 18,000 × g for 5 minutes, and the supernatant was used as an electrophoresis sample.
[0038]
Using the marker and sample prepared above, electrophoresis was performed on an acrylamide gel prepared previously.
After the electrophoresis, the gel was removed from the gel plate, placed in a container containing the reaction solution, and shaken at 30 ° C. for 5 minutes. After repeating the same operation twice, the gel was immersed in the reaction solution and incubated at 30 ° C. for 120 minutes. The reaction solution was discarded, an iodine solution was added, and the mixture was shaken for about 15 minutes to stain the gel. After staining, observation was performed using an illuminator.
FIG. 13 shows the results.
[0039]
Example 6 Porphyridium purpurum R-1 (P. purpurum R-1)
Isolation of isoamylase (ISA) cDNA clone
(1) Preparation of ISA probe
Using primers (forward primers, F1, and F2; reverse primers, R1, and R2) prepared based on the amino acid sequence of a region (conserved region) commonly found in the ISA gene (see FIG. 14), PCR was performed using cDNA of R. purpureum R-1 (P. purpureum) as a template, and the obtained PCR fragment was sequenced. Since the amino acid sequence predicted from the obtained nucleotide sequence showed homology to that of ISA of other organisms, a 68-mer probe was prepared based on the nucleotide sequence (sequence between Box I and II) of this portion. .
[0040]
(2) Primary screening
E. coli (XL1-blue qLI-blue MRF 'strain) cultured overnight in LB medium (OD ₆₀₀ ) Was measured and centrifuged at 1,500 × g for 10 minutes. ₆₀₀ = 0.5mM MgSO ₄ And suspended. To 1.5 ml of this E. coli suspension was added 8 μl of a phage solution (10,000 clones), and the mixture was incubated at 37 ° C. for 15 minutes.
After the incubation, 600 μl of phage-infected Escherichia coli was added to the soft agar and quickly spread on a plate (150 mM) medium kept in a 37 ° C. incubator. The mixture was allowed to stand at room temperature for 15 minutes to harden the software. The solidified plate medium was inverted and left standing for 6-8 hours in a 37 ° C incubator, and then stored at 4 ° C.
Membrane (Hybond-N ⁺ , Amersham Pharmacia) was gently placed on the plate medium so as to prevent air bubbles from entering, and allowed to stand for 1 minute. Marks were pierced at three places with a syringe needle with ink. The membrane was peeled off with tweezers. This operation was performed for two membranes per one plate. The membrane was allowed to stand for 7 minutes on a filter paper impregnated with a denaturing solution with the surface in contact with the plate medium facing up. After standing, the membrane was placed on dry filter paper, and left on the filter paper impregnated with the neutralizing solution for 3 minutes. This operation was repeated twice. The membrane was immersed in 2 × SSC buffer and washed for about 1 minute. The membrane was placed on a dry filter paper, dried and heated at 80 ° C. for 2 hours.
Prehybridization and hybridization were performed using the probe (labeled with a radioactive element) prepared above. Rapid-hyb buffer (Amersham Pharmacia) was used for pre-hybridization and hybridization. The membrane was immersed in Rapid-hyb buffer warmed at 42 ° C and shaken at 42 ° C for 30 minutes to perform prehybridization.
The X-ray film was developed and examined for the presence of positive plaque.
[0041]
(3) Secondary screening
The resulting positive plaque was cut from the plate and 500 μl of SM buffer (5.8 g of NaCl in 1 L, MgSO 4) ₄ ・ 7H ₂ O 2.0 g, 1M Tris-HCl (pH 7.5) 50.0 ml, 2% gelatin 5.0 ml) was placed in an Eppendorf tube, and one drop of chloroform was added. After vortexing, it was stored at 4 ° C.
Escherichia coli was cultured in an LB medium in the same manner as in the primary screening, and centrifuged at 1,500 × g for 10 minutes. ₆₀₀ = 0.5mM MgSO ₄ Was added and suspended. OD ₆₀₀ To 200 μl of the host cell of 0.5, 1 μl of the phage stock solution diluted to 1/100, 1/1000 or 1 / 10,000 with SM buffer was added, and the mixture was incubated at 37 ° C. for 15 minutes. After the incubation, Escherichia coli infected with the phage was added to the top of 3 ml, and spread on an NZY plate (90 mM). The top was allowed to set for 10 minutes at room temperature, and the plate was inverted and incubated at 37 ° C. overnight to obtain a plaque. As in the primary screening, two hybridization membranes were prepared for each plate.
The subsequent experimental operation was performed in the same manner as in the primary screening until development.
[0042]
(4) In vivo Excision
Positive plaques were cut from the plate, placed in an Ebbendorf tube containing 500 μl of SM buffer, and one drop of chloroform was added. After vortexing, it was stored at 4 ° C. XL1-blue MRF 'strain and SOLR strain were added to LB medium in 0.2% maltose and 10 mM MgSO4. ₄ Were cultivated overnight at 30 ° C. in a medium supplemented with. Centrifuge at 1,000 xg for 10 minutes and OD ₆₀₀ = 10 mM MgSO ₄ Was added and suspended. XL1-blue MRF 'strain (OD ₆₀₀ = 1.0) 200 μl, 250 μl of phage stock and 1 μl of EX Assist helper phage were placed in a tube and incubated at 37 ° C. for 15 minutes. 3 ml of LB medium was added, and shaking culture was performed at 37 ° C. for 2 to 3 hours. After the culture, the mixture was heated at 65 to 70 ° C. for 20 minutes and centrifuged at 1,000 μg for 15 minutes. The supernatant was decanted to a new tube. When used as stock, it was stored at 4 ° C. When performing plating, use SOLR cells (OD ₆₀₀ = 1.0) 100 μl or 10 μl of the phage supernatant was added to 200 μl, and the mixture was incubated at 37 ° C. for 15 minutes. 200 μl of each cell mixture was spread on an LB-ampicillin agar plate and cultured at 37 ° C. overnight.
[0043]
(4) Extraction of plasmid
The colony obtained in the above (3) was picked with a toothpick, planted on a medium containing 2 × YT medium and ampicillin, and cultured at 37 ° C. overnight. After centrifugation at 3,000 × g for 1 minute, the supernatant was discarded. Add 200 μl of Solution I (50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0)), mix by pipetting, add 300 μl of Solution II (0.2 N NaOH, 1% SDS) and add tube Was mixed by inversion and allowed to stand on ice for 5 minutes. 300 μl of solution III (3M potassium acetate (pH 4.8)) was added, the tube was inverted, mixed, allowed to stand on ice for 5 minutes, centrifuged at 3,000 × g, room temperature for 10 minutes, and the supernatant was transferred to a new tube. 2 μl of 10 mg / ml RNase A was added to the supernatant, and the mixture was allowed to stand at 37 ° C. for 20 minutes. After adding 400 μl of chloroform and rolling by hand for 30 seconds, the mixture was centrifuged for 1 minute, and the aqueous layer (upper layer) was transferred to a new tube. This operation was performed twice. 600 μl of 2-propanol was added to the aqueous layer, and the mixture was centrifuged at 18,000 × g at room temperature for 10 minutes. The supernatant was discarded, and 500 μl of 70% ethanol was added to the precipitate. Centrifugation was repeated once again, the supernatant was discarded, and the precipitate was dried with an aspirator for about 10 minutes. After drying, 33.6 μl of sterile water, 6.4 μl of 5M NaCl, and 40 μl of 13% PEG6000 were added, and the mixture was stirred well and allowed to stand on ice for 20 minutes. This was centrifuged at 18,000 × g at 4 ° C. for 15 minutes, and the supernatant was carefully discarded. The precipitate was washed with 500 μl of 70% ethanol and dried with an aspirator. The obtained DNA was dissolved by adding 10 μl of sterilized water, ₂₆₀ , A ₂₈₀ Was measured to calculate the DNA concentration and stored at -20 ° C.
[0044]
(5) PCR for checking length of cDNA clone
To examine the length of the cDNA portion (insert) of the cDNA clone, PCR was performed using each clone as a template.
[0045]
Example 7 Determination of base sequence of ISA gene
3.2 μl of M13 primer F or R (1 pmol / μl), 4 μl of Big Dye premix, 4 μl of dilution buffer, and 20 μl of distilled water for template DNA (0.25-0.5 μg) PCR was performed under the following conditions.
After the PCR, 2 μl of 3M sodium acetate and 50 μl of 95% ethanol were added to the PCR product, and the mixture was left at room temperature for 15 minutes. After standing, the mixture was centrifuged at 18,000 × g for 20 minutes, carefully sucked without leaving the supernatant, and 800 μl of 70% ethanol was added to the precipitate, followed by gentle stirring. The mixture was centrifuged at 18,000 × g for 5 minutes to remove the supernatant, and dried with an aspirator. 20 μl of HiDi formamide was added, suspended, heated at 95 ° C. for 2 minutes, ice-cooled, and subjected to a sequencer.
The obtained base sequence is shown in SEQ ID NO: 1. The amino acid sequence predicted from the obtained nucleotide sequence is shown in SEQ ID NO: 2. FIG. 15 shows a part of the predicted amino acid sequence.
[0046]
【The invention's effect】
The present invention provides a novel isoamylase and a gene encoding the same. The isoamylase of the present invention is derived from red algae, which has an intermediate structure between starch produced by higher plants such as rice and phytoglycogen produced by cyanobacteria. By combining the isoamylase of the above with a starch synthase of a higher plant, starches having a novel cluster structure can be produced, which may provide a new useful starch for food or industry.
[0047]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the starch chain length distribution of Porphyridium purpureum R-1. The horizontal axis in FIG. 1 shows the chain length (number of glucose (DP)), and the vertical axis shows the ratio (%) of the amount of each chain length to the total amount of the total chain length.
FIG. 2 shows the starch chain length distribution of Galdieria sulphuraria M-8. The horizontal axis in FIG. 2 shows the chain length (number of glucose (DP)), and the vertical axis shows the ratio (%) of the amount of each chain length to the total amount of the total chain length.
FIG. 3 shows the starch chain length distribution of Cyanidium caldarium N-551. The horizontal axis in FIG. 3 shows the chain length (number of glucose (DP)), and the vertical axis shows the ratio (%) of the amount of each chain length to the total amount of the total chain length.
FIG. 4 shows the starch chain length distribution of Gelidium (Tengsa). The horizontal axis in FIG. 4 indicates the chain length (number of glucose (DP)), and the vertical axis indicates the ratio (%) of the amount of each chain length to the total amount of the total chain length.
FIG. 5 shows the starch chain length distribution of rice (Nipponbare). The horizontal axis in FIG. 5 indicates the chain length (number of glucose (DP)), and the vertical axis indicates the ratio (%) of the amount of each chain length to the total amount of the total chain length.
FIG. 6 is a color photograph instead of a drawing showing the result of a color reaction by iodine starch reaction of each fraction of gel filtration chromatography of P. purpurumum.
FIG. 7 shows the results of gel filtration chromatography of the red alga P. purpureum. FIG. 7 (a) (upper) shows the measurement based on the absorbance at 595 nm in the iodine starch reaction, and FIG. 7 (b) (lower) shows the measurement based on the quantitative determination of reducing sugars by the phenol-sulfuric acid method. In each case, the sample was 20 mg, the flow rate was 0.14 mL / min, and Sephacryl S-100 was used as the column. The horizontal axis of (a) and (b) of FIG. 7 shows the fraction number, the vertical axis of (a) shows the absorbance of each fraction at 595 nm, and the vertical axis of (b) shows the absorbance of each fraction at 490 nm. ing.
FIG. 8 shows the determination of reducing sugars of the red alga Gardieria Sulfuraria by the phenol-sulfuric acid method. The sample was 25 mg, the flow rate was 0.16 mL / min, and the column was Sephacryl S. -100 (Sephacryl S-100) was used. The horizontal axis in FIG. 8 shows the fraction number, and the vertical axis shows the absorbance at 490 nm of each fraction.
FIG. 9 shows green alga C. Fig. 9 shows the results of gel filtration chromatography of reinhardtii. In FIG. 9, (a) (upper row) is a measurement based on the absorbance at 595 nm in the iodine starch reaction, and (b) (lower row) is a measurement based on the quantification of reducing sugar by the phenol-sulfuric acid method. In each case, the sample was 20 mg, the flow rate was 0.14 mL / min, and Sephacryl S-100 was used as the column. In FIG. 9, the horizontal axes of (a) and (b) indicate the fraction number, the vertical axis of (a) indicates the absorbance of each fraction at 595 nm, and the vertical axis of (b) indicates the absorbance of each fraction at 490 nm. Is shown.
FIG. 10 shows green alga C. 2 shows the results of gel filtration chromatography of Kessleri. In FIG. 10, (a) (upper) is a measurement based on the absorbance at 595 nm in the iodine starch reaction, and (b) (lower) is a measurement based on the quantification of reducing sugar by the phenol-sulfuric acid method. In each case, the sample was 20 mg, the flow rate was 0.14 mL / min, and Sephacryl S-100 was used as the column. In FIG. 10, the horizontal axes of (a) and (b) indicate the fraction numbers, the vertical axis of (a) indicates the absorbance of each fraction at 595 nm, and the vertical axis of (b) indicates the absorbance of each fraction at 490 nm. Is shown.
FIG. 11 shows the results of gel filtration chromatography of rice (Fujihikari). In FIG. 11, (a) (upper) is a measurement based on the absorbance at 595 nm in the iodine starch reaction, and (b) (lower) is a measurement based on the quantification of reducing sugar by the phenol-sulfuric acid method. In each case, the sample was 20 mg, the flow rate was 0.14 mL / min, and Sephacryl S-100 was used as the column. In each of FIG. 11, the horizontal axis of (a) and (b) indicates the fraction number, the vertical axis of (a) indicates the absorbance of each fraction at 595 nm, and the vertical axis of (b) indicates the 490 nm of each fraction. The absorbance is shown.
FIG. 12 shows the results of analyzing the chain length distribution of the fractions of amylopectin and amylose in the fractions obtained by gel filtration chromatography of Porphyridium purpurium (P. purpureum) and rice (Fujihikari). (A) (upper) of FIG. 12 is a comparison of the amylopectin fraction, and (b) (lower) is a comparison of the amylose fraction. The gray bars in FIG. 12 indicate rice (Fujihikari), and the black bars indicate the red alga Porphyridium purpurium. 12A and 12B, the horizontal axis indicates the chain length (number of glucose (DP)), and the vertical axis indicates the ratio (%) of the amount of each chain length to the total amount of the total chain length.
FIG. 13 is a photograph instead of a drawing showing the results of testing the ISA activity of P. purpureum by activity staining (Native PAGE) using an iodine starch reaction. FIG. 13 Lanes 1 and 2 are ripening seed extracts of rice as a marker, and Lane 3 is a crushed liquid obtained by crushing cells of a red alga Porphyridium purpureum (P. purpureum) with glass beads. 4 is the supernatant of centrifugation at 18,000 × g. The left side of FIG. 13 shows the position of pullulanase (PUL), and the right side shows the position of isoamylase (ISA).
FIG. 14 shows regions (conserved regions) commonly found in the ISA gene. FIG. 14 illustrates two types of maize (maize endosperm), rice (rice endosperm), and cyanobacteria (Synechocystis). The lower part of FIG. 14 shows PCR primers for ISA of Red Alga of the present invention.
FIG. 15 shows a part of the predicted amino acid sequence of the isoamylase (ISA) of the present invention in one-letter amino acid notation.
FIG. 16 shows a comparison of the amino acid sequences of ISA of various organisms and the ISA of the present invention. FIG. 16 shows the amino acid sequence of the portion of the red alga Porphyridium purpurium of the present invention that could be decoded from the top, and the lower row shows the rice ISA (Rice-ISA). The column shows the ISA of cyanobacterial glgX (Scysis-glgX), the lower column shows the ISA of cyanobacterial glgXX (Scysis-glgXX), and the lower column shows the ISA of Pseudomonas amyloderamosa (P. amylo-ISA). Show.
FIG. 17 is a continuation of FIG. 16 showing a comparison of the amino acid sequences of ISAs of various organisms and the ISA of the present invention.
FIG. 18 shows a phylogenetic tree prepared by adding the sequence of ISA from another organism to the sequence of ISA from clone 1 obtained by the method of the present invention. The numbers above each branch in FIG. 18 indicate a boot strap.

Claims (10)

Isoamylase derived from Porphyridium, a kind of red algae. The isoamylase according to claim 1, wherein the isoamylase partially has the amino acid sequence of SEQ ID NO: 2 in the sequence listing. A gene encoding the isoamylase according to claim 1. The gene according to claim 3, wherein the gene is DNA. The gene according to claim 4, wherein the gene has a base sequence of SEQ ID NO: 1 in a part thereof. An oligonucleotide having at least 10 contiguous bases of the gene according to claim 3. The oligonucleotide according to claim 6, wherein the base is at least 15 bases or more. A vector comprising the gene according to any one of claims 3 to 5. The vector according to claim 8, wherein the vector is an expression vector. A transformant into which the gene according to any one of claims 3 to 5 has been introduced.

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