JP4257933B2 - Isoamylase from the red alga Porphyridium - Google Patents

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]
BACKGROUND OF THE INVENTION
The present invention relates to 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 consisting of a partial sequence of a base sequence of a gene encoding isoamylase derived from a kind of algae, Porphyridium.
The isoamylase of the present invention is a kind of enzyme that synthesizes starch specific to red algae. By utilizing the enzyme activity, it is possible to develop starch for food or industry having a novel structure. It becomes possible.
[0002]
[Prior art]
Starch is not only used as a main ingredient of grains as food and feed, but also processed into dextrins, oligosaccharides, isomerized sugars, etc., and is also used in processed foods. It is also used as a raw material. Even if it is simply called starch, rice starch, potato starch, wheat starch, corn starch, etc., depending on its origin, the shape, taste, and physical properties when gelatinized are slightly different. Different types of plant-derived starch have been used depending on the application. It has been explained that the difference in the properties of starch is caused by the difference in the chemical structure of starch, but starch is generally made of amylose and amylopectin, and the difference in chemical structure is mainly branched structure. It is said that the place derived from amylopectin having
Starch is an α-polyglucan that accumulates in higher plant storage organs. Starch consists 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 glucoside bonds and contains a small amount of α-1,6 glucoside bond branches. On the other hand, amylopectin has a structure in which a glucose unit extends with α-1,4 glucoside bonds and branches extend from the main chain with α-1,6 glucoside bonds. This branching forms a cluster and forms a characteristic structure called a “cluster” structure.
Glycogen, which is a storage material for animals and bacteria, is composed of glucose homopolymers as well as amylopectin, but it does not have a cluster structure and has a random structure called “tree like” or “bush like”. have. Glycogen consists of a completely irregular branched structure. Glycogen has smaller molecules and shorter branches than amylopectin, and many of them are water-soluble substances. In contrast, amylopectin has a long branch and is filled with glucose at a high density, and is generally a water-insoluble substance.
[0003]
The outline of starch synthesis is as follows. (1) Starch synthase (SS), (2) Starch branching enzyme (SBE), (3) Starch debranching enzyme (Starch) synthesized by the reaction of debranching enzyme (DBE)). SS has the role of extending the chain by linking ADP glucose to the non-reducing end of amylopectin via an α-1,4 glucoside bond. While SS extends the amylopectin chain, SBE is an enzyme that forms an α-1,6 glucoside bond of amylopectin, and an enzyme that forms a branched portion of a branched structure. Traditionally, starch debranching enzyme (DBE) was thought to be an enzyme that is not required for cluster formation. However, it has been clarified that a plant lacking this degrading enzyme cannot form an 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]
Thus, in order to biosynthesize amylopectin, which is a component of starch, the presence of isoamylase, which is a kind of DBE, is indispensable. Therefore, higher plants that synthesize starch have isoamylases according to each plant. . Organisms that do not produce starch, such as animals, do not have isoamylase, but some organisms do not produce starch but have isoamylase. The reason for the presence of isoamylase in such organisms is not clear at present, and future research is expected. To date, the cyanobacterial isoamylase (ISA) gene has been isolated (see Patent Document 1), but no isoamylase has been isolated for other primitive organisms.
[0005]
Red algae are photosynthetic organisms born by the coexistence of cyanobacteria with eukaryotic cells that are the ancestors of red algae, and are said to be one of the oldest eukaryotes. The chloroplasts of red algae are derived from green plants, but genetically no affinity with green plants is observed. This is considered to be a result of a host that is genetically unrelated to the green plant taking in the green plant and coexisting with it. Red algae is an organism that has a great deal of interest both in terms of organism evolution and taxonomics.
There are marine and freshwater red algae, and it is known that they are extremely diverse from microalgae to macroalgae. Red algae are taxonomically classified as organisms belonging to the Rhodophyceae group of the Eukaryophyta, and about 600 genera and 5000 species are currently known. About 200 species are known as fresh water.
[0006]
However, there is little information on the starch synthesis and metabolic system of red algae, and research on the metabolism of carbohydrates in red algae has been conducted since the 1960s. At that time, mainly the red alga starch of the large alga Dilsea edulis, and Fredrick et al. (1971) were conducting research on the isozyme of Cyanidium caldarium, a primitive red algae (see Non-Patent Document 4). At this time, the starch of red algae has red starch algae (floridean Starch) peculiar to red algae from the color, viscosity and average chain length of iodine starch reaction, and it has no amylose structurally, only amylopectin It was said to be a composition. However, in the 1980s, the presence of amylopectin and amylose was suggested in the starch of the red alga Glaucosphaera vacuolata (see Non-Patent Document 5). Since then, red algae starch is divided into two types: starch of the primitive red algae (Protoflorideophycidae), which is thought to have amylose in the starch granules, and starch of the true red algae (Florideophycidae) containing red algae starch. It became so. However, there are reports that the primitive red algae Cyanidiosizon merolae and C. caldarium have a phytoglycogen-like starch structure, and the starch structure of the red algae has not been clearly clarified. In order to develop a starch having a novel amylopectin structure, it is required to clarify the starch structure of red algae and the enzyme system thereof.
[0007]
[Patent Document 1]
JP 2002-262877 A
[Non-Patent Document 1]
MGJames, et al., Plant Physiol., (1995) 7, 417-429
[Non-Patent Document 2]
Y. Nakamura, et al., PlantJ., (1997) 12 (1), 143-153
[Non-Patent Document 3]
A. Kubo, et al., Plant Phys., (1999) 121, 399-409
[Non-Patent Document 4]
Fredrick, JF, Phytochemistry, (1971) 10, 395-398
[Non-Patent Document 5]
McCracken, DA, et al., New Phytol., (1981) 88, 67-71
[0008]
[Problems to be solved by the invention]
The object of the present invention is to analyze the structure of starch of red algae and to isolate and identify its synthase. Red algae starch has a unique structure different from other organisms, and it is not only useful as a new starch for food and industrial use, but also its synthase has 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 the structural analysis of starch of red algae, and identified the isoamylase, which led to the present invention.
[0010]
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 gene, and a transformant containing the gene.
Furthermore, the present invention relates to a partial sequence of the base sequence of the gene, more specifically, an oligonucleotide comprising at least 10 consecutive bases.
[0011]
The present inventors, as biological materials, are Porphyridium purpureum R-1 (Galdieria sulphuraria M-8) belonging to the genus Protoflorideophycidae. , Cyanidium caldarium N-551, Porphyra (Porphyra), and Geridium (Gelidium) belonging to the true red algae subclass (Florideophycidae) The structure of the starch was analyzed, and a novel isoamylase was successfully isolated from Porphyridium purpureum R-1.
[0012]
The present inventors cultured these organisms for several days, centrifuged the cells, disrupted the cells, and centrifuged the resulting cell disruption solution 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 higher plant rice (Nipponbare).
Porphyridium purpureum R-1, Galdieria sulphuraria M-8, Cyanidium caldarium N-551, and Gelidium (Gelidium ( FIG. 1 to FIG. 5 show the results of the starch chain length analysis for the rice (Tengusa) and rice (Nipponbare), respectively. 1 to 5, the horizontal axis represents the chain length (glucose number (DP)), and the vertical axis represents the ratio (%) of the amount of each chain length to the total amount of all chain lengths. Show. FIG. 1 is a starch of Porphyridium purpureum R-1, FIG. 2 is a starch of Galdieria sulphuraria M-8, and FIG. 3 is a cyanidium starch. Fig. 4 shows the starch of Calidium N-551, Fig. 4 shows the starch of Gelidium, and Fig. 5 shows the starch of Ricebare.
[0013]
As a result, there is a chain between G. sulphuraria, C. caldarium and P. purpureum, despite the same primitive red alga subclass. There was a difference in the length distribution. Of these, G. sulphuraria and C. caldarium have many short chain lengths below DP10 and are considered to be close to the chain length distribution of cyanobacteria. However, a prominent peak is seen in the vicinity of DP8, which may contain impurities such as oligosaccharides. On the other hand, the chain length distribution of P. purpureum is similar to that of Gelidium of the true red algae subclass, both of which have a shorter chain length than rice and have a lower peak of DP10-15 chain length. The peak of DP45, which is considered necessary for forming, was lower than that of rice.
[0014]
Next, gel filtration chromatography of starch was performed on red alga P. purpureum, G. sulphuraria, green algae Chlamydomonas reinhardtii CC125, Chlorella kesleri 11h, and higher plant rice (Fujihikari). It was. For the red alga P. purpureum, green algae C. reinhardtii and C. kessleri, the starch obtained by the Percoll method is used, and for the red alga G. sulphuraria Since the starch was not obtained by the (Percoll) method, the starch obtained by the DMSO extraction method was used.
The result of the color reaction by the iodine starch reaction of each fraction of the red alga Porphyridium purpureum (P. purpureum) is shown in FIG. Fraction 11-13 showed a purple coloration and fractions 19-25 showed a blue coloration. FIG. 7 shows the results of gel filtration chromatography of the red alga Porphyridium purpureum (P. purpureum). (A) (upper part) of FIG. 7 is a measurement by the absorbance at 595 nm in the iodine starch reaction, and (b) (lower part) is a measurement by quantitative determination 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 (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 at 595 nm of each fraction, and the vertical axis of (b) shows the absorbance at 490 nm of each fraction. ing.
As can be seen from this figure, two peaks were obtained from the measurement of absorbance at 595 nm, and λmax in the respective peak fractions (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. Moreover, the light absorbency of 490 nm by the coloration by the phenol sulfuric acid method was measured (refer FIG.7 (b)). The graph of 595 nm and the graph of the phenol-sulfuric acid method almost coincide with each other considering that amylose is 20 times more likely to be stained than amylopectin. A comparison of the amount of reducing sugars between amylopectin and amylose based on the results of the phenol-sulfuric acid method showed that amylopectin was considerably abundant.
[0015]
The red alga G. sulphuraria was not colored by iodine starch reaction. This may be because the starch in G. sulphuraria has a structure that is difficult to be stained with iodine, or because there was not enough amount to allow staining with iodine. The amount of reducing sugar was thought to be small because no coloration was observed due to iodine starch reaction, and in the phenol sulfuric acid method, the reaction was carried out with 50 μl of each fraction and 10 μl of 0.5 M HCl, unlike normal conditions. . As a result, a single peak was obtained. The results are shown in FIG. FIG. 8 shows the measurement of the red alga G. sulphuraria by quantitative determination of reducing sugars by the phenol-sulfuric acid method. The sample is 25 mg, the flow rate is 0.16 mL / min, and the column is Sephacryl S-100 (Sephacryl S-100). -100) was used. The horizontal axis in FIG. 8 indicates the fraction number, and the vertical axis indicates the absorbance at 490 nm of each fraction.
This suggests that, unlike the starch of P. purpureum containing amylopectin and amylose, the starch of G. supHuraria is composed of a single component.
[0016]
Similarly, gel filtration chromatography was performed on starches of green algae C. reinhardtii and C. kessleri and rice starch.
FIG. 9 shows the results of gel filtration chromatography of the green alga C. reinhardtii. FIG. 10 shows the results of gel filtration chromatography of the green alga C. kessleri. FIG. 11 shows the results of gel filtration chromatography of rice (Fujihikari).
In each of FIGS. 9 to 11, (a) (upper) is a measurement by absorbance at 595 nm in the iodine starch reaction, and (b) (lower) is a measurement by 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 (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 at 595 nm of each fraction, and the vertical axis of (b) indicates the fraction of each fraction. Absorbance at 490 nm is shown.
[0017]
The starches of green algae C. reinhardtii and C. kessleri had amylopectin and amylose, similar to rice starch (see FIGS. 9 (a) and 10 (a)). Comparing the results of the phenol-sulfuric acid method of these green algae with that of the red alga P. purpureum, the green algae had a slightly higher amylose content (C. reinhardtii 22.4%, C. kessleri). 13.0%, P. purpureum (6.4%) (see FIG. 9B and FIG. 10B). In rice, the iodine starch reaction and the phenol-sulfuric acid method both have a high amylose peak and a large content compared to red algae and green algae (amylose content 29.4%) ((a) and (b) in FIG. 11). reference).
Table 1 shows the results obtained by measuring the λmax near the peak obtained from the iodine starch reaction and averaging it to give a standard error.
[0018]
[Table 1]

[0019]
Next, the chain length distributions of fractions of amylopectin and amylose obtained from gel filtration chromatography of P. purpureum and rice (Fujihikari) were analyzed. Both chain length distributions are shown in FIG. (A) (upper part) of FIG. 12 is a comparison of the amylopectin fraction, and (b) (lower part) is a comparison of the amylose fraction. The gray bars in FIG. 12 indicate rice (Fujihikari), and the black bars indicate red alga Porphyridium purpureum (P. purpureum). 12 (a) and 12 (b), 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 P. purpureum, the amylose fraction had a slightly larger amount of short difference compared to the amylopectin fraction, but 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. The same was true for Fujihikari in this regard.
In addition, the amylopectin of P. purpureum has more short chains of DP8 or less than Fujihikari, but the distribution up to around DP10-25 was less. There was also a difference in the vicinity of DP45, with a gentle peak in Fujihikari, but no peak in P. purpureum.
[0020]
From the above, starch of red alga P. purpureum (P. purpureum) has many short chains compared to rice, and long chain around DP45, which is thought to be necessary to form a cluster structure. It can be seen that the peaks are low and that there are few long chains. Cyanobacteria are known to have a large number of short chains of DP10 or less mainly, and have no peak near DP45, which is considered necessary for forming a cluster structure. Green algae has a short chain distribution. Although there is a slight difference, it shows a chain length distribution similar to that of higher plant rice, and a peak in the vicinity of DP45 is also observed, indicating that a cluster structure is formed. There is a slight difference in short chains between rice and green algae, but the long chain peak around DP45 is commonly observed, so green algae form a starch structure closer to higher plants than red algae. It is thought that. Since cyanobacteria synthesize phytoglycogen, in chain length analysis, there are many short chains up to the vicinity of DP3-8, and many do not have long chains after DP30.
Therefore, although P. purpureum has fewer short chains than cyanobacteria, long chains are found after DP30. From these facts, it is considered that the starch of P. purpureum is an intermediate type of cyanobacteria and rice. Structurally, it is not as strong as the cluster structure of rice amylopectin, but it is thought that it is not as branched as the phytoglycogen of cyanobacteria. On the other hand, the red alga C. caldarium and G. sulphuraria, which have different chain length distributions from P. purpureum, have very short chains and run orchids. It is thought that the chain length distribution is close to that of algae.
The large alga Gelidium has few side chains near DP10-15, but otherwise it is very similar to the chain length analysis of P. purpureum, so Gelidium starch Is thought to contain structures more similar to those of P. purpureum than those of C. caldarium and G. sulphuraria.
[0021]
Thus, the existence of red algae with or without amylose was revealed. In particular, in G. sulphuraria, although the presence of reducing sugar was confirmed by the phenol-sulfuric acid method, no color was obtained in the iodine starch reaction. In addition, when comparing the elution position of the gel filtration chromatography of starch of P. purpureum (P. purpureum), it was eluted from around 40 ml in P. purpureum, In the case of G. sulphuraria, it was eluted from around 80 ml. For these reasons, the polyglycan of G. sulphuraria is composed of one component, is not easily 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 structure is considered to be an incomplete amylopectin structure or a phytoglycogen-like structure, and it is considered that it has a phytoglycogen-like structure when the results of chain length analysis are added. It was also found that both red algae and green algae are seldom less amylose than rice.
[0022]
In higher plants, isoamylase (ISA) is considered essential for the formation of starch cluster structures. Therefore, the presence of ISA was examined in the red alga P. purpureum, which was confirmed to have a starch cluster structure.
FIG. 13 shows a photograph in place of a drawing of the result of testing the activity of ISA of porphyridium purpureum by activity staining (Native PAGE) using iodine starch reaction. Fig. 13 Lanes 1 and 2 are ripening seed extracts of rice as a marker, and lane 3 is a crushed solution obtained by crushing cells of the red alga P. purpureum with glass beads. 4 is the supernatant obtained by 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, the presence of ISA was also confirmed in the red alga P. purpureum (see lanes 3 and 4 in FIG. 13). From this, it is considered that ISA is involved in the synthesis or degradation of amylopectin even in P. purpureum like green plants.
In this activity staining, the amylopectin in the gel is easily stained with iodine when the α-1,6 glycosidic bond is cleaved by ISA and the number of branches decreases, but when it is cleaved with amylase, it becomes glucose. It is broken down into sugars such as maltose and oligosaccharides, so it is not stained with iodine. Therefore, some bands appearing white in FIG. 13 are observed, but this is considered to be amylase.
[0023]
Red algae are considered to have a unique starch metabolism system, such as the presence of UDP-glucose as a glucose donor and the presence or absence of amylose synthesis. Therefore, among the enzymes related to starch synthesis, the ISA cDNA that could be confirmed by the above experiment was first cloned and its base sequence was determined, and compared with cyanobacteria, green algae and higher plants, We decided to examine whether there was a difference.
[0024]
ISA is known to have a region (conserved region) commonly found in ISA genes. This is shown in FIG. Two examples are maize (maize endosperm), rice (rice endosperm) and cyanobacteria (Synechocystis). All have storage areas in the four areas I, II, III, and IV shown in FIG.
Therefore, PCR was carried out using a red alga P. purpureum cDNA as a template using a primer prepared based on the amino acid sequence of a region (conserved region) commonly found in the ISA gene. . When PCR was performed with various combinations of primers, PCR was performed with F1 and R1 primers, and nested PCR was performed with F1 and R2 primers (shown in the lower part of FIG. 14) using the PCR product as a template. As a result, a PCR product of the expected size was obtained. The obtained PCR fragment was sequenced. Since the amino acid sequence predicted from the obtained base sequence showed homology with that of ISA of other organisms, a 68-mer probe was prepared based on the base sequence of this part (sequence between Box I and II). .
[0025]
Using this probe, about 5 × 10 ⁵ When the clones were screened, 5 positive clones were obtained in the primary screening, and when the secondary screening was performed, positive clones were obtained for all 5 types. Of the 5 positive clones, 2 were successful in in vivo excision, and 4 were in addition to those with different dilutions during in vivo excision. It was. Two pieces were taken from the plate and a total of 8 pieces were applied to the sequence. As a result of the sequencing, 3 clones having different sequences were obtained except for 8 clones from which similar sequences were obtained.
Among the 3 clones obtained, clone 2 was highly homologous to the DegP protein (AY039585-1) of Arabidopsis thaliana from homology search, so the analysis was finished.
Clone 1 and clone 3 contained sequences that matched the sequence of the PCR product described above.
The base sequence that can be decoded from these clones is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence expected to be encoded by the gene is shown in SEQ ID NO: 2. FIG. 15 shows an amino acid sequence represented by one letter code for amino acids.
[0026]
The homology of the base sequences of clone 1 and clone 3 was 99.4%, and the amino acid sequences deduced from the base sequences of these clones showed high homology with those of ISAs of other organisms. FIG. 16 and FIG. 17 show a comparison of amino acid sequences of ISA of various organisms and ISA of the present invention. FIGS. 16 and 17 show the amino acid sequence of the decipherable portion of the red alga P. purpureum of the present invention from the top, and the lower row shows the rice ISA (Rice-ISA). The lower stage shows ISA (Scystis-glgX) of cyanobacteria glgX, the lower stage shows ISA (Scystis-glgXX) of cyanobacteria glgXX, and the lower stage shows ISA (P. amyloramosa) of Pseudomonas amyloderamosa. -ISA). FIG. 17 is a continuation of FIG.
Further, FIG. 18 shows a phylogenetic tree prepared by adding the ISA sequences of other organisms to the clone 1 sequence. The numbers on each branch in FIG. 18 indicate the boot strap.
[0027]
The present invention provides an isoamylase derived from the genus Porphyridium of the genus Porphyridiales of the primordial red algae subfamily of red algae, and more particularly an isoamylase derived from Purphyridium purpureum, and a gene encoding the same. Is. The present invention confirms the presence of isoamylase from red algae and clones the gene encoding the enzyme. Although the complete analysis of the base sequence has not been completed, the method described herein is used. The desired red algae gene can be obtained, and its base sequence can be decoded by a conventional method.
Although the isoamylase of the present invention is derived from R-1 of P. purpureum in the above-described example, it is not limited to this.
As described above, the isoamylase of the present invention can form a structure that is structurally as firm as the cluster structure of rice amylopectin, such as an intermediate form of cyanobacteria and rice, which is a starch unique to red algae. Although not, it is considered useful for the production of starch having a structure that is not as randomly branched as phytoglycogens of cyanobacteria. In general, starch with a poor cluster structure is more water-soluble than starch with a firm structure, and has characteristics such as gelatinization temperature that are important for producing completely new starch. It is considered an enzyme.
The isoamylase of the present invention is assumed to have a partial amino acid sequence (see SEQ ID NO: 2), but is not limited to those having this amino acid sequence, and is not limited to red algae, preferably Porphyridiales, More preferably, it is derived from the genus Porphyridium, more preferably from the genus Purphyridium purpureum, and it may be any as long as it has an activity capable of forming starch unique to the aforementioned red algae.
[0028]
The isoamylase of the present invention can be produced by the above-described method, but is not limited to this method, and a known method such as a PCR method is combined using the base sequence provided by the present invention. Can be produced by various methods.
The gene cloning method of the present invention is not limited to the above-described method, and various known cloning methods can be used.
Furthermore, the gene of the isoamylase of the present invention can be used by connecting genes having various base sequences upstream and / or downstream if necessary. In any case, the gene of the present invention As long as it contains, it belongs to the technical scope of the present invention. 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. As the promoter, the red alga P. purpureum can be used, but other organisms can also be combined.
[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 shown in the above example, or an expression vector to which a promoter is bound. Various plasmids can be selected as the 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. If the host cell already has an isoamylase gene, it may be knocked out, or the gene of the present invention may be introduced as it is. Cells in which the gene of the present invention has been introduced and expressed in this manner are subjected to starch debranching by the activity of the isoamylase of the present invention, and production of starch having a structure similar to that of red alga starch is performed. 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 probe for cloning, a probe for detecting the presence of the gene of the present invention, and the like. Although there is no restriction | limiting in particular in the length of the oligonucleotide of this invention, Preferably 10-150 bases, 15-150 bases, 20-150 bases, 15-50 bases, etc. can be used suitably.
[0031]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
Biological materials such as Porphyridium purpureum R-1 (Chinorimo), Galdieria sulphuraria M-8, and Cyanidium caldarium N-551 551) were obtained from the IAM Culture Collection of the Institute for Molecular Cytology, the University of Tokyo. Sorbinori (Porphyra sp.) Was obtained from the Uchikaijo Functional Education Research Center, Kobe University. Tengusa (Gelidium sp.) Was collected from Ehime beach.
P. purpureum is MKM medium at 20 ° C, 2% CO ₂ G. sulphuraria was cultured in Allen medium at 33 ° C with air bubbling, and C. caldarium was cultured in A15L medium at 33 ° C with air bubbling. did.
[0032]
Example 1 (Extraction of starch)
(1) Extraction from small organisms
Cells such as P. purpureum cultured for several days were centrifuged at 1,700 × g, 4 ° C. for 10 minutes. Methanol was added and vortexed with glass beads for 10 minutes to disrupt the cells. After centrifugation at 1,700 × g and 4 ° C. for 10 minutes, methanol was added again to the precipitate, and the above operation was repeated until the dye was removed. Dimethyl sulfoxide (DMSO) was added to the centrifuged precipitate and boiled at 90 ° C. for 120 minutes. After boiling, the mixture was transferred to a 50 ml tube, centrifuged at 1,700 × g, 4 ° C. for 10 minutes, the supernatant was placed in a 1 L medium bottle, DMSO was added to the precipitate again, and the mixture was boiled at 90 ° C. for 120 minutes. After boiling, the above centrifugation operation was performed once again. The obtained supernatant was put into a 1 L medium bottle, and about 800 ml of ethanol was added, and 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 some grinding, methanol was added and the mixture was stirred at -20 ° C overnight. The next day, the mixture was centrifuged at 2,000 × g for 10 minutes, the precipitate was put 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. Subsequent operations were performed in the same manner as starch extraction of microalgae.
[0034]
Example 2 (Analysis by capillary electrophoresis)
Aggregates of α-1,4 glucans of various lengths obtained by completely degrading starch α-1,6 glycosidic bonds with isoamylase were obtained. ATPS (1-aminopyrene-3,6,8-trisulfonic acid trisodium salt), which is a fluorescent substance, was added to the reducing ends of all α-1,4 glucans obtained by enzyme treatment, and they were electrophoresed to obtain 488 nm. Highly sensitive detection was performed 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 (Structural analysis of starch by gel filtration chromatography)
(1) Sample preparation
The microalgal starch was suspended by adding 1 ml of ethanol, centrifuged at 10,000 × g, 4 ° C., 10 minutes, the supernatant was removed, and dried with an aspirator.
For rice (Fujihikari) starch, the endosperm was placed in a mortar and ground with a pestle, ethanol was suspended, and the mixture was centrifuged 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 and suspended, and 0.4 ml of 5N NaOH was added and mixed. The starch was gelatinized by leaving it in a 47 ° C. water bath for 30 minutes. 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
The 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).
Sixty preparative test tubes (6 ml) were set in the fraction collector 1 (ATTO). The flow rate was about 0.14 ml / min, and about 4 ml was fractionated per test tube.
The result of the color reaction by the iodine starch reaction of each fraction of the obtained gel filtration chromatography of P. purpureum is shown in FIG.
Moreover, the result of each gel filtration chromatography is shown in FIGS.
[0036]
Example 4 (Chain length analysis of each fraction in gel filtration)
Among the fractions obtained by gel filtration chromatography, the fractions estimated to be amylopectin and amylose from the measurement of λmax were collected in medium bottles, ethanol was added, and the starch was precipitated overnight at −20 ° C. The starch was collected by centrifugation, and each was analyzed for chain length by capillary electrophoresis as described above.
The results are shown in FIG.
[0037]
Example 5 (Activity staining of starch synthesis / degradation related enzyme (Native PAGE))
In order to investigate the enzyme activity related to 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 and mixed with 3.3 ml of distilled water, covered with a wrap, and melted at 60 ° C. with a stirrer until clear. 30% acrylamide, 0.8% bis-acrylamide, 1.5M Tris-HCl (pH 8.8), 0.2% TEMED, 1.65 ml each, and amylopectin solution 3.3 ml were mixed to obtain 10% AMPS (ammonium persulfate). 30 μl 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. 30% acrylamide, 0.8% bis-acrylamide, 1.0 M Tris-HCl (pH 6.8), 0.2% TEMED each 0.3 ml, distilled water 1.8 ml and mixed, APS 16 μl and gel Poured into a plate, pointed to 10 or 12 lane combs and left for 20 minutes.
Rice (ripening) straw and embryos were removed and placed in an Eppendorf tube, and the weight of endosperm was measured. Cell disruption buffer (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 plastic pestle. Centrifugation was performed at 18,000 × g and 4 ° C. for 20 minutes, and the supernatant was placed in another tube. This supernatant was mixed with the sample buffer so as to have a ratio of 2: 1 to obtain an electrophoresis marker.
Sample cells 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 put into a test tube containing glass beads, vortexed for 10 minutes to destroy the cells, 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 the previously prepared acrylamide gel.
After 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 the same operation was repeated 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 gel was stained by shaking for about 15 minutes. After dyeing, observation was performed with an illuminator.
The results are shown in FIG.
[0039]
Example 6 Isolation of Isoamylase (ISA) cDNA Clone of P. purpureum R-1
(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), Porphy PCR was performed using the cDNA of L. purpureum R-1 (P. purpureum) as a template, and the resulting PCR fragment was sequenced. Since the amino acid sequence predicted from the obtained base sequence showed homology with that of ISA of other organisms, a 68-mer probe was prepared based on the base sequence of this part (sequence between Box I and II). .
[0040]
(2) Primary screening
The concentration (OD1) of E. coli (XL1-blue qLI-blue MRF 'strain) cultured overnight in LB medium ₆₀₀ ), And centrifuged at 1,500 × g for 10 minutes, and the concentration of E. coli is OD ₆₀₀ = 10 mM MgSO so that 0.5 ₄ Suspended in To 1.5 ml of this Escherichia coli suspension, 8 μl of phage solution (10,000 clones) was added and incubated at 37 ° C. for 15 minutes.
After incubation, 600 μl of E. coli infected with phages was added to soft agar and spread quickly on a plate (150 mM) medium kept warm at 37 ° C. incubator. It was allowed to stand at room temperature for 15 minutes to harden the software. The solidified plate medium was inverted and allowed to stand for 6 to 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 that no air bubbles would enter it, and allowed to stand for 1 minute. Three piercing marks were made with an injection needle with ink. The membrane was peeled off with tweezers. This operation was performed on two membranes per plate. The membrane was allowed to stand for 7 minutes on a filter paper soaked with the denaturing solution with the surface in contact with the plate medium facing up. After standing, the membrane was placed on a dry filter paper, and left on the filter paper soaked 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 dry filter paper, dried, and heated at 80 ° C. for 2 hours.
Prehybridization and hybridization were performed using the probe prepared above (labeled with a radioactive element). A rapid-hyb buffer (Amersham Pharmacia) was used for prehybridization and hybridization. The membrane was immersed in Rapid-hyb buffer warmed at 42 ° C. and shaken at 42 ° C. for 30 minutes for prehybridization.
The X-ray film was developed and examined for the presence of positive plaque.
[0041]
(3) Secondary screening
The resulting positive plaque was excised from the plate and 500 μl SM buffer (5.8 g NaCl, MgSO4 in 1 L). ₄ ・ 7H ₂ A drop of chloroform was added to an eppendorf tube containing 2.0 g of O, 1M Tris-HCl (pH 7.5) 50.0 ml, 2% gelatin 5.0 ml). After vortexing, it was stored at 4 ° C.
As in the first screening, E. coli is cultured in LB medium, centrifuged at 1,500 × g for 10 minutes, and OD is added to the precipitate. ₆₀₀ = 10 mM MgSO so that 0.5 ₄ And suspended. OD ₆₀₀ = 200 μl of 0.5 host cells, 1 μl of a phage stock solution diluted to 1/100, 1/1000 or 1 / 10,000 in SM buffer was added and incubated at 37 ° C. for 15 minutes. After incubation, E. coli infected with phage was added to the top of 3 ml and spread on NZY plates (90 mM). The top was set for 10 minutes at room temperature, the plate was inverted and incubated overnight at 37 ° C. to obtain a plaque. Similar to the primary screening, two hybridization membranes were prepared for each plate.
Subsequent experimental operations were performed in the same manner as the primary screening until development.
[0042]
(4) In vivo Excision
Positive plaques were cut from the plates and placed in an Ebendorf tube containing 500 μl SM buffer and a 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 with 0.2% maltose and 10 mM MgSO. ₄ In each of the above mediums, the cells were cultured overnight at 30 ° C. 1,000 xg, centrifuge for 10 minutes, OD ₆₀₀ = 10 mM MgSO so that = 10 ₄ And suspended. XL1-blue MRF 'strain (OD ₆₀₀ = 1.0) 200 μl, phage stock 250 μl and EX Assist helper phage 1 μl 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 incubation, the mixture was heated at 65 to 70 ° C. for 20 minutes and centrifuged at μ1,000 × g for 15 minutes. The supernatant was transferred to a new tube by decantation. When used as a stock, it was stored at 4 ° C. When plating, use SOLR cells (OD ₆₀₀ = 1.0) 100 μl or 10 μl of phage supernatant was added to 200 μl and incubated at 37 ° C. for 15 minutes. 200 μl of each cell mixture was plated on LB-ampicillin agar plates and cultured at 37 ° C. overnight.
[0043]
(4) Plasmid extraction
The colony obtained in (3) above was picked with a toothpick and planted in a medium containing 2 × YT medium and ampicillin and cultured at 37 ° C. overnight. The mixture was centrifuged at 3,000 × g for 1 minute, and 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)) and mix by pipetting, add 300 μl of Solution II (0.2 N NaOH, 1% SDS) and invert the tube to mix. And left 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 at 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 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 centrifuged at 18,000 × g for 10 minutes at room temperature. 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 sterilized water, 6.4 μl of 5M NaCl, and 40 μl of 13% PEG6000 were added and stirred well, and left on ice for 20 minutes. This was centrifuged at 18,000 × g for 15 minutes at 4 ° C., the supernatant was carefully discarded, 500 μl of 70% ethanol was added to the precipitate, washed and dried with an aspirator. 10 μl of sterilized water is added to the obtained DNA and dissolved. ₂₆₀ , A ₂₈₀ Was measured to calculate the DNA concentration and stored at -20 ° C.
[0044]
(5) PCR for examining the length of cDNA clones
In order 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 nucleotide sequence of ISA gene
For template DNA (0.25-0.5 μg), M13 primer F or R (1 pmol / μl) is 3.2 μl, Big Dye premix is 4 μl, dilution buffer is 4 μl, and distilled water is 20 μl in total. In addition, PCR was performed under the following conditions.
After PCR, 2 μl of 3M sodium acetate and 50 μl of 95% ethanol were added to the PCR product and left at room temperature for 15 minutes. After standing, the mixture was centrifuged at 18,000 × g for 20 minutes, carefully sucked out so as not to leave a supernatant, and 800 μl of 70% ethanol was added to the precipitate and stirred gently. The supernatant was removed by centrifugation at 18,000 × g for 5 minutes and dried with an aspirator. 20 μl of HiDi formamide was added, suspended, heated at 95 ° C. for 2 minutes, ice-cooled, and applied to a sequencer.
The obtained base sequence is shown in SEQ ID NO: 1. The amino acid sequence predicted from the obtained base sequence is shown in SEQ ID NO: 2. A part of the predicted amino acid sequence is shown in FIG.
[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 above isoamylase with starch synthase from higher plants, starches having a novel cluster structure can be produced, and there is a possibility that new useful starches for food and industry can be provided.
[0047]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the chain length distribution of starch of Porphyridium purpureum R-1. The horizontal axis in FIG. 1 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 all chain lengths.
FIG. 2 shows the chain length distribution of starch of Galdieria sulphuraria M-8. The horizontal axis of 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 all chain lengths.
FIG. 3 shows the chain length distribution of starch of Cyanidium caldarium N-551. The horizontal axis in FIG. 3 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 all chain lengths.
FIG. 4 shows the chain length distribution of the gelidium (Gelidium) starch. The horizontal axis of FIG. 4 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 all chain lengths.
FIG. 5 shows the chain length distribution of Nipponbare starch. 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 all chain lengths.
FIG. 6 is a color photograph instead of a drawing showing the results of a color reaction by iodine starch reaction of each fraction of gel filtration chromatography of P. purpureum.
FIG. 7 shows the results of gel filtration chromatography of the red alga P. purpureum. (A) (upper part) of FIG. 7 is a measurement by the absorbance at 595 nm in the iodine starch reaction, and (b) (lower part) is a measurement by quantitative determination 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 (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 at 595 nm of each fraction, and the vertical axis of (b) shows the absorbance at 490 nm of each fraction. ing.
FIG. 8 shows the measurement of the red alga G. sulphuraria by quantification of reducing sugars by the phenol-sulfuric acid method, the sample is 25 mg, the flow rate is 0.16 mL / min, and the column is Sephacryl S. -100 (Sephacryl S-100) was used. The horizontal axis in FIG. 8 indicates the fraction number, and the vertical axis indicates the absorbance at 490 nm of each fraction.
FIG. 9 shows the results of gel filtration chromatography of the green alga C. reinhardtii. In FIG. 9, (a) (upper part) is a measurement by absorbance at 595 nm in the iodine starch reaction, and (b) (lower part) is a measurement by quantitative determination 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 (Sephacryl S-100) was used as the column. In FIG. 9, the horizontal axis of (a) and (b) indicates the fraction number, the vertical axis of (a) indicates the absorbance at 595 nm of each fraction, and the vertical axis of (b) indicates the absorbance at 490 nm of each fraction. Show.
FIG. 10 shows the results of gel filtration chromatography of the green alga C. kessleri. (A) (upper part) of FIG. 10 is a measurement by absorbance at 595 nm in the iodine starch reaction, and (b) (lower part) is a measurement by quantitative determination 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 (Sephacryl S-100) was used as the column. In FIG. 10, the horizontal axis of (a) and (b) indicates the fraction number, the vertical axis of (a) indicates the absorbance at 595 nm of each fraction, and the vertical axis of (b) indicates the absorbance at 490 nm of each fraction. Show.
FIG. 11 shows the results of gel filtration chromatography of rice (Fujihikari). (A) (upper part) of FIG. 11 is a measurement by absorbance at 595 nm in the iodine starch reaction, and (b) (lower part) is a measurement by quantitative determination 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 (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 at 595 nm of each fraction, and the vertical axis of (b) indicates the 490 nm of each fraction. Absorbance is shown.
FIG. 12 shows the results of analyzing the chain length distribution of fractions of amylopectin and amylose in fractions obtained by gel filtration chromatography of P. purpureum and rice (Fujihikari). (A) (upper part) of FIG. 12 is a comparison of the amylopectin fraction, and (b) (lower part) is a comparison of the amylose fraction. The gray bars in FIG. 12 indicate rice (Fujihikari), and the black bars indicate red alga Porphyridium purpureum (P. purpureum). 12 (a) and 12 (b), 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 replacing a drawing which shows the result of testing the activity of P. purpureum ISA by activity staining using iodine starch reaction (Native PAGE). Fig. 13 Lanes 1 and 2 are ripening seed extracts of rice as a marker, and lane 3 is a crushed solution obtained by crushing cells of the red alga P. purpureum with glass beads. 4 is the supernatant obtained by 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 a region (conserved region) 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 the red alga ISA of the present invention.
FIG. 15 shows a part of the predicted amino acid sequence of the isoamylase (ISA) of the present invention by one-letter code of amino acid.
FIG. 16 shows a comparison of the amino acid sequences of ISA of various organisms and ISA of the present invention. From the top of FIG. 16, the amino acid sequence of the decipherable portion of the red alga P. purpureum of the present invention is shown, the lower row shows the rice ISA (Rice-ISA), The stage shows the ISA of cyanobacterium glgX (Scystis-glgX), the lower stage shows the ISA of cyanobacterium glgXX (Scystis-glgXX), and the lower stage 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 the ISA of various organisms and the ISA of the present invention.
FIG. 18 shows a phylogenetic tree prepared by adding the ISA sequences of other organisms to the ISA sequences from clone 1 obtained by the method of the present invention. The numbers on each branch in FIG. 18 indicate the boot strap.

Claims (8)

An isoamylase derived from Porphyridium , a kind of red algae, having a portion of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. A gene encoding the isoamylase according to claim 1 . The gene according to claim 2 , wherein the gene is DNA. The gene according to claim 3 , wherein the gene has a base sequence described in SEQ ID NO: 1 in the sequence listing. An oligonucleotide having at least 20 consecutive bases of the gene according to any one of claims 2 to 4 . A vector comprising the gene according to any one of claims 2 to 4 . The vector according to claim 6 , wherein the vector is an expression vector. A transformant into which the gene according to any one of claims 2 to 4 has been introduced.

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