JP3557271B2 - DNA encoding an enzyme, recombinant DNA containing the same, and transformant - Google Patents

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【００３９】
【表２】

【００４０】
表１及び表２の結果から明らかなように、酵素Ｍ−１１及び酵素Ｑ３６は、マルトトリオース、マルトテトラオース、マルトペンタオース、マルトヘキサオース及びマルトヘプタオースなどのグルコース重合度が３以上の還元性澱粉糖からは新たな糖質を生成するけれども、グルコース重合度が３を下回るグルコースやマルトースからは新たな糖質を生成しない。また、反応により生成した糖質はそれぞれ糖質Ｐ１乃至Ｐ５のみであり、糖質Ｐ２乃至Ｐ５の含量は固形分当たり８５％以上と著しく高かった。
【００４１】
次に、糖質Ｐ１乃至Ｐ５を分離すべく、東京有機化学工業製強酸性カチオン交換樹脂『ＸＴ−１０１６（Ｎａ^＋型）』を内径２．０ｃｍ、長さ１ｍのジャケッ ト付きステンレス製カラム３本に充填し、これらカラムを直列に連結した。そして、カラム内の温度を５５℃に保ちつつ、カラムに糖質Ｐ１乃至Ｐ５のいずれかを含む前記反応物を別々に負荷した後、カラムに５５℃の蒸留水をＳＶ０．１３の流速で通液した。溶出液の糖組成を調べ、糖質Ｐ１乃至Ｐ５のいずれかを固形分で９７％以上含む画分を採取し、真空乾燥により粉末化した。このようにして精製した糖質Ｐ１乃至Ｐ５の還元力をソモギ・ネルソン法により調べたところ、いずれの糖質にも実質的な還元力は認められなかった。
【００４２】
さらに、糖質Ｐ１乃至Ｐ５を同定すべく、これら糖質のいずれかを５０ｍｇとり、５０ｍＭ酢酸緩衝液（ｐＨ４．５）１ｍｌに溶解後、グルコアミラーゼを１単位加え、４０℃で６時間インキュベートした。表１及び表２に示す反応物の糖組成を高速液体クロマトグラフィーにより分析したところ、それぞれ表３及び表４に示すように、全ての反応物からグルコースとトレハロースが検出された。同様にして、糖質Ｐ１乃至Ｐ５にβ−アミラーゼを作用させたところ、糖質Ｐ１及びＰ２がβ−アミラーゼの作用を受けなかったのに対して、糖質Ｐ３は１分子のマルトースと糖質Ｐ１を、糖質Ｐ４は１分子のマルトースと糖質Ｐ２を、また、糖質Ｐ５は２分子のマルトースと糖質Ｐ１を与えた。
【００４３】
【表３】

【００４４】
【表４】

【００４５】
表３及び表４の結果は、糖質Ｐ１乃至Ｐ５が１分子のトレハロースと１乃至５分子のグルコースにより構成されることを強く示唆している。また、グルコアミラーゼがマルトオリゴ糖におけるα−１，４結合及びα−１，６結合に特異的に切断することと、β−アミラーゼがマルトオリゴ糖におけるα−１，４結合をその末端よりマルトース単位で切断することから、糖質Ｐ１乃至Ｐ５は、グルコース又はグルコース重合度が２乃至５のマルトオリゴ糖の末端にトレハロース残基が１個結合した構造を有していると推定される。
【００４６】
以上の結果を総合的に判断すると、糖質Ｐ１乃至Ｐ５は、それぞれ、α−グルコシルトレハロース、α−マルトシルトレハロース、α−マルトトリオシルトレハロース、α−マルトテトラオシルトレハロース又はα−マルトペンタオシルトレハロースと同定され、このことは、当該酵素にグルコース重合度３以上の還元性澱粉糖から末端にトレハロース構造を有する非還元性糖質を生成する作用のあることを裏付けている。
【００４７】
【実験例２−２ 分子量】
ユー・ケー・レムリが『ネーチャー』、第２２７巻、第６８０〜６８５頁（１９７０年）に報告している方法に準じて精製酵素をＳＤＳ−ポリアクリルアミドゲル電気泳動したところ、酵素Ｍ−１１、酵素Ｑ３６とも、分子量約７６，０００乃至８７，０００ダルトンに相当する位置に単一バンドが観察された。なお、このときの分子量マーカは、ミオシン（２００，０００ダルトン）、β−ガラクトシダーゼ（１１６，２５０ダルトン）、フォスフォリラーゼＢ（９７，４００ダルトン）、血清アルブミン（６６，２００ダルトン）及びオボアルブミン（４５，０００ダルトン）であった。
【００４８】
【実験例２−３ 等電点】
等電点電気泳動法により測定したところ、酵素Ｍ−１１、酵素Ｑ３６とも、約３．６乃至４．６に等電点を示した。
【００４９】
【実験例２−４ 至適温度】
常法により、５０ｍＭ燐酸緩衝液（ｐＨ７．０）中で６０分間インキュベートする条件で試験したところ、図１又は図２に示すように、酵素Ｍ−１１、酵素Ｑ３６とも、３５乃至４０℃付近に至適温度を示した。
【００５０】
【実験例２−５ 至適ｐＨ】
常法により、ｐＨの相違する５０ｍＭ酢酸緩衝液、燐酸緩衝液又は炭酸ナトリウム−炭酸水素ナトリウム緩衝液中、４０℃で６０分間インキュベートする条件で試験したところ、図３又は図４に示すように、酵素Ｍ−１１、酵素Ｑ３６とも、ｐＨ６．４乃至７．２付近に至適ｐＨを示した。
【００５１】
【実験例２−６ 熱安定性】
常法により、５０ｍＭ燐酸緩衝液（ｐＨ７．０）中で６０分間インキュベートする条件で試験したところ、図５又は図６に示すように、酵素Ｍ−１１、酵素Ｑ３６とも、３５乃至４０℃付近まで安定であった。
【００５２】
【実験例２−７ ｐＨ安定性】
常法により、ｐＨの相違する５０ｍＭ酢酸緩衝液、燐酸緩衝液又は炭酸ナトリウム−炭酸水素ナトリウム緩衝液中、２５℃で１６時間インキュベートする条件で試験したところ、図７又は図８に示すように、酵素Ｍ−１１、酵素Ｑ３６とも、ｐＨ５．５乃至１１．０付近まで安定であった。
【００５３】
【実験例２−８ Ｎ末端アミノ酸配列】
常法により、アプライッド・バイオシステム製気相プロテイン・シーケンサ『４７０Ａ型』を使用して分析したところ、酵素Ｍ−１１は、Ｎ末端に配列表における配列番号７に示すアミノ酸配列を有していることが判明した。
【００５４】
同様に分析したところ、酵素Ｑ３６は、Ｎ末端に配列表における配列番号８に示すアミノ酸配列を有していることが判明した。
【００５５】
【実験例２−９ 部分アミノ酸配列】
実験例１−１で得た精製酵素Ｍ−１１を適量とり、１０ｍＭトリス−塩酸緩衝液（ｐＨ９．０）に対して４℃で１８時間透析後、１０ｍＭトリス−塩酸緩衝液（ｐＨ９．０）を加えて酵素濃度を約１ｍｇ／ｍｌとした。この溶液を約１ｍｌとり、リジルエンドペプチダーゼを１０μｇ加え、３０℃で２２時間インキュベートして酵素を部分加水分解した。加水分解物を、予め１６％（ｖ／ｖ）水性アセトニトリルを含む０．１％（ｖ／ｖ）トリフルオロ酢酸により平衡化させておいた資生堂製逆相高速液体クロマトグラフィー用カラム『カプセルパックＣ１８』に負荷し、次いで、１６％（ｖ／ｖ）から６４％（ｖ／ｖ）に上昇するアセトニトリルの濃度勾配下、カラムに０．１％（ｖ／ｖ）トリフルオロ酢酸を０．９ｍｌ／分の流速で通液した。そして、通液開始から約２８分後又は約４０分後に溶出したペプチド断片（以下、それぞれ「ペプチド断片Ａ」又は「ペプチド断片Ｂ」と云う。）を含む画分を別々に採取し、真空乾燥後、５０％（ｖ／ｖ）水性アセトニトリルを含む０．１％（ｖ／ｖ）トリフルオロ酢酸に溶解した。以後、実験例２−８と同様に分析したところ、ペプチド断片Ａ及びＢは、配列表における配列番号９及び１０に示すアミノ酸配列を有していることが判明した。
【００５６】
別途、実験例１−２で得た精製酵素Ｑ３６を上記と同様にして部分加水分解し、予め２４％（ｖ／ｖ）水性アセトニトリルを含む０．１％（ｖ／ｖ）トリフルオロ酢酸により平衡化させておいた日本ミリポア・リミテッド製逆相高速液体クロマトグラフィー用カラム『マイクロボンダパックＣ１８』に負荷し、２４％（ｖ／ｖ）から４４％（ｖ／ｖ）に上昇する水性アセトニトリルの濃度勾配下、カラムに０．１％（ｖ／ｖ）トリフルオロ酢酸を０．９ｍｌ／分の流速で通液した。そして、通液開始から約２２分後又は約４０分後に溶出したペプチド断片（以下、それぞれ「ペプチド断片Ｃ」又は「ペプチド断片Ｄ」と云う。）を含む画分を採取し、真空乾燥後、５０％（ｖ／ｖ）水性アセトニトリルを含む０．１％（ｖ／ｖ）トリフルオロ酢酸に溶解した。以後、上記と同様に分析したところ、ペプチド断片Ｃ及びＤは、配列表における配列番号１１及び１２に示すアミノ酸配列を有していることが判明した。
【００５７】
酵素Ｍ−１１と酵素Ｑ３６の理化学的性質は以上のとおりであるが、以下の実施例では、これら理化学的性質に基づき、この発明による複製可能な組換えＤＮＡと形質転換体を調製するとともに、そこに含まれている当該酵素をコードするＤＮＡを採取し、その塩基配列とアミノ酸配列を決定する。なお、これら実施例で用いる手法自体は斯界において公知のものであり、例えば、ジェー・サムブルック等『モレキュラー・クローニング・ア・ラボラトリー・マニュアル』、第２版、１９８９年、コールド・スプリング・ハーバー・ラボラトリー・プレス発行などにも詳述されている。
【００５８】
【実施例１ リゾビウム・スピーシーズＭ−１１に由来するＤＮＡを含む組換えＤＮＡと形質転換体の調製】
【００５９】
【実施例１−１ 染色体ＤＮＡの調製】
リゾビウム・スピーシーズＭ−１１をバクト・ニュートリエント・ブロス培地（ｐＨ７．０）に植菌し、２７℃で２４時間回転振盪培養した。遠心分離により培養物から菌体を分離し、ＴＥＳ緩衝液（ｐＨ８．０）に浮遊させ、リゾチームを０．０５％（ｗ／ｖ）加えた後、３７℃で３０分間インキュベートした。処理物を−８０℃で１時間凍結後、ＴＳＳ緩衝液（ｐＨ９．０）を加えて６０℃に加温し、ＴＥＳ緩衝液／フェノール混液を加え、氷冷後、遠心分離により上清を採取した。この上清に２倍容の冷エタノールを加え、沈澱した粗染色体ＤＮＡを採取し、ＳＳＣ緩衝液（ｐＨ７．１）に溶解後、リボヌクレアーゼとプロテアーゼをそれぞれ７．５μｇ又は１２５μｇ加え、３７℃で１時間インキュベートして反応させた。その後、反応物にクロロフォルム／イソアミルアルコール混液を加えて染色体ＤＮＡを抽出し、冷エタノールを加え、生成した染色体ＤＮＡを含む沈澱を採取した。このようにして得た精製染色体ＤＮＡを濃度約１ｍｇ／ｍｌになるようにＳＳＣ緩衝液（ｐＨ７．１）に溶解し、溶液を−８０℃で凍結した。
【００６０】
【実施例１−２ 組換えＤＮＡ ｐＢＭＴ７と形質転換体ＢＭＴ７の調製】実施例１−１で得た精製染色体ＤＮＡ溶液を約１ｍｌとり、これに制限酵素Ｓａｕ ３ＡＩを約３５単位加え、３７℃で約２０分間反応させて染色体ＤＮＡを部分切断した後、蔗糖密度勾配超遠心法により約３，０００乃至７，０００塩基対からなるＤＮＡ断片を採取した。別途、プラスミドベクターＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）を１μｇとり、常法により制限酵素Ｂａｍ ＨＩを作用させて完全に切断した後、上記で得たＤＮＡ断片１０μｇとＴ４ ＤＮＡリガーゼを２単位加え、４℃で一夜静置することによりＤＮＡ断片をベクター断片に連結した。そして、得られた組換えＤＮＡに東洋紡績製コンピテントセル『Ｅｐｉｃｕｒｉａｎ Ｃｏｌｉ ＸＬＩ−Ｂｌｕｅ』を３０μｌ加え、氷冷下に３０分間静置後、４２℃に加温し、ＳＯＣブロスを加えて３７℃で１時間インキュベートすることにより、組換えＤＮＡを大腸菌に導入した。
【００６１】
次に、上記で得た形質転換体を５−ブロモ−４−クロロ−３−インドリル−β−ガラクトシド５０μｇ／ｍｌを含む寒天平板培地（ｐＨ７．０）に植菌し、３７℃で１８時間培養後、培地上にナイロン膜を載置し、培地上に形成された約４，４００個のコロニーをナイロン膜に固定した。別途、常法により、配列表における配列番号９に示すアミノ酸配列における第１７乃至２１番目のＰｒｏ−Ｇｌｕ−Ｔｒｐ−Ｇｌｕ−Ｌｙｓで表される配列に基づき５′−ＣＣＮＧＡＲＴＧＧＧＡＲＡＡ−３′で表される塩基配列のプローブ１を化学合成し、同位体^３２Ｐで標識後、前記ナイロン膜上に固定した形質転換体のコロニーにハイブリダイズさせ、顕著な会合が認められた９種類の形質転換体を選択した。
【００６２】
常法により、これら９種類の形質転換体から組換えＤＮＡを採取し、配列表における配列番号１０に示すアミノ酸配列における第１６乃至２０番目のＴｈｒ−Ｇｌｕ−Ｐｈｅ−Ｔｒｐ−Ａｓｐで表される配列に基づき化学合成した５′−ＡＣＮＧＡＲＴＴＹＴＧＧＧＡ−３′で表される塩基配列のプローブ２をイー・エム・サザーン『ジャーナル・オブ・モレキュラー・バイオロジー』、第９８巻、第５０３〜５１７頁（１９７５年）に記載されている方法に準じてハイブリダイズさせ、プローブ２と顕著な会合を示した組換えＤＮＡを選択した。以上のようにして選択した組換えＤＮＡと形質転換体を、それぞれ、『ｐＢＭＴ７』又は『ＢＭＴ７』と命名した。
【００６３】
上記で得た形質転換体ＢＭＴ７をアンピシリン１００μｇ／ｍｌを含むＬ−ブロス培地（ｐＨ７．０）に植菌し、３７℃で２４時間回転振盪培養した。培養終了後、遠心分離により培養物から菌体を採取し、通常一般のアルカリ法により組換えＤＮＡを菌体外に溶出させた。処理物を常法により精製し、分析したところ、組換えＤＮＡ ｐＢＭＴ７は約９，３００塩基対からなり、図９に示す制限酵素地図で表される構造を有していた。図９に示すように、酵素Ｍ−１１をコードする２，３１６塩基対からなるＤＮＡは、制限酵素Ｐｓｔ Ｉによる切断部位付近の下流に位置していることが判明した。
【００６４】
【実施例１−３ 形質転換体ＢＭＴ７による酵素の産生】
マルトース２．０％（ｗ／ｖ）、ペプトン０．５％（ｗ／ｖ）、酵母エキス０．１％（ｗ／ｖ）、燐酸水素二ナトリウム０．１％（ｗ／ｖ）、燐酸二水素カリウム０．１％（ｗ／ｖ）を含む液体培地をｐＨ７．０に調整し、アンピシリンを５０μｇ／ｍｌ加え、１２０℃で２０分間加熱滅菌し、冷却後、実施例１−２で得た形質転換体ＢＭＴ７を植菌し、３７℃で２４時間回転振盪培養した。培養物を超音波処理して菌体を破砕し、遠心分離により不溶物を除去後、上清中の酵素活性を測定したところ、培養物１ｌ当たりに換算して、約３，０００単位の酵素が産生していた。
【００６５】
別途、対照として、大腸菌ＸＬＩ−Ｂｌｕｅ株及びリゾビウム・スピーシーズＭ−１１をアンピシリン無含有の同じ液体培地に植菌し、リゾビウム・スピーシーズＭ−１１の場合、培養温度を３０℃に設定した以外は上記と同様に培養・処理した。処理物の活性を測定したところ、リゾビウム・スピーシーズＭ−１１による酵素の産生は培養物１ｌ当たり約１，５００単位と、形質転換体ＢＭＴ７と比較して有意に低いものであった。なお、宿主に使用した大腸菌ＸＬＩ−Ｂｌｕｅ株は、当該酵素を全く産生しなかった。
【００６６】
その後、形質転換体ＢＭＴ７が産生した酵素を実験例１−１と同様に精製し、その性質・性状を調べたところ、ＳＤＳ−ポリアクリルアミドゲル電気泳動で分子量値約７６，０００乃至８７，０００ダルトンを、また、等電点電気泳動で約３．６乃至４．６に等電点を示すなど、実験例２で得られた酵素Ｍ−１１のものと同様の理化学的性質を有することが判明した。このことは、組換えＤＮＡ技術によっても当該酵素を製造でき、且つ、酵素の生産性も有意に向上することを示唆している。
【００６７】
【実施例２ リゾビウム・スピーシーズＭ−１１に由来する相補鎖ＤＮＡの調製とその塩基配列、アミノ酸配列の決定】
実施例１−２で得た組換えＤＮＡ ｐＢＭＴ７を、常法に従って、各種制限酵素で分解し、Ｂｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）にサブクローニングして、塩基配列決定用ＤＮＡとした。これら塩基配列決定用ＤＮＡを２μｇとり、これに２Ｍ水酸化ナトリウム水溶液を加えて変性させた後、適量の冷エタノールを加え、生成したテンプレートＤＮＡを含む沈澱を採取し、真空乾燥した。このテンプレートＤＮＡに化学合成した５′−ＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧＴ−３′で表される塩基配列のプライマー１を５０ｐｍｏｌ／ｍｌと、２０ｍＭ塩化マグネシウムと５０ｍＭ塩化ナトリウムを含む４０ｍＭトリス−塩酸緩衝液（ｐＨ７．５）を１０μｌ加え、６５℃で２分間インキュベートしてアニーリングした後、ｄＡＴＰ、ｄＧＴＰ及びｄＴＴＰをそれぞれ７．５μＭ含む水溶液を２μｌと、［α−^３２Ｐ］ｄＣＴＰ（２ｍＣｉ／ｍｌ）を０．５μｌと、０．１Ｍジチオスレイトールを１μｌと、１．５単位／ｍｌのＴ７ ＤＮＡポリメラーゼを２μｌ加え、２５℃で５分間インキュベートすることによりプライマー１を５′末端から３′末端に向かって伸長させ、相補鎖ＤＮＡを生成させた。
【００６８】
次に、上記で得た相補鎖ＤＮＡを含む反応物を四等分し、それぞれにｄｄＡＴＰ、ｄｄＣＴＰ、ｄｄＧＴＰ及びｄｄＴＴＰのいずれかを８μＭと８０μＭ ｄＮＴＰを含む５０ｍＭ塩化ナトリウム水溶液を２．５μｌ加え、３７℃で５分間インキュベートして反応させた後、２０ｍＭ ＥＤＴＡ、０．０５％（ｗ／ｖ）ブロムフェノールブルー及び０．０５％（ｗ／ｖ）キシレンシアノールを含む９５％（ｖ／ｖ）水性ホルムアミド溶液を４μｌ加えて反応を停止させた。反応物を沸騰水浴中で３分間加熱後、６％（ｗ／ｖ）ポリアクリルアミドゲル上にとり、約２，０００Ｖの定電圧を印加しながら電気泳動してＤＮＡ断片を分離し、次いで、常法によりゲルを固定し、乾燥させた後、オートラジオグラフィーした。
【００６９】
ラジオグラム上に分離したＤＮＡ断片を解析した結果、相補鎖ＤＮＡは配列表における配列番号５に示す２，９３６塩基対からなる塩基配列を含んでいることが判明した。この塩基配列から推定されるアミノ酸配列は配列表における配列番号５に併記したとおりであり、このアミノ酸配列と配列表における配列番号７、９又は１０に示す酵素Ｍ−１１のＮ末端アミノ酸配列、部分アミノ酸配列を比較したところ、配列番号７のＮ末端アミノ酸配列は配列表における配列番号５における第１乃至２０番目の配列に、また、配列番号９又は１０の部分アミノ酸配列は配列表における配列番号５における第４８６乃至５０６番目又は第６０６乃至６２６番目の配列に一致した。これは、酵素Ｍ−１１が配列表における配列番号１に示すアミノ酸配列を有するものであり、リゾビウム・スピーシーズＭ−１１においては、酵素Ｍ−１１が配列表における配列番号３に示す塩基配列のＤＮＡによりコードされていることを示している。
【００７０】
【実施例３ アルスロバクター・スピーシーズＱ３６に由来するＤＮＡを含む組換えＤＮＡと形質転換体の調製】
【００７１】
【実施例３−１ 染色体ＤＮＡの調製】
実施例１−１と同様にしてアルスロバクター・スピーシーズＱ３６から染色体ＤＮＡを分離・精製し、濃度約１ｍｇ／ｍｌになるようにＳＳＣ緩衝液（ｐＨ７．１）に溶解し、−８０℃で凍結した。
【００７２】
【実施例３−２ 組換えＤＮＡ ｐＢＱＴ１３と形質転換体ＢＱＴ１３の調製】
実施例３−１で得た精製染色体ＤＮＡ溶液を実施例１−２と同様に部分切断した後、蔗糖密度勾配超遠心法により約３，０００乃至６，０００塩基対からなるＤＮＡ断片を採取した。その後、Ｔ４ ＤＮＡリガーゼを使用し、このＤＮＡ断片を実施例１−２と同様に制限酵素Ｂａｍ ＨＩによるベクターＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）の消化物に連結し、得られた組換えＤＮＡを大腸菌ＸＬＩ−Ｂｌｕｅ株に導入した。得られた形質転換体を実施例１−２と同様に５−ブロモ−４−クロロ−３−インドリル−β−ガラクトシドを含む寒天平板培地で培養し、生成した約４，５００個のコロニーをナイロン膜上に固定する一方、配列表における配列番号１２に示すアミノ酸配列における第１１乃至１６番目のＰｈｅ−Ａｓｐ−Ｖａｌ−Ａｓｐ−Ｔｒｐ−Ａｓｐで表される配列に基づき５′−ＴＴＹＧＡＹＧＴＮＧＡＹＴＧＧＧＡ−３′で表される塩基配列のプローブ３を化学合成し、同位体^３２Ｐで標識後、前記ナイロン膜上に固定した形質転換体のコロニーにハイブリダイズさせ、顕著な会合が認められた８種類の形質転換体を選択した。
【００７３】
実施例１−２と同様にして、これら８種類の形質転換体から組換えＤＮＡを採取し、配列表における配列番号１１に示すアミノ酸配列における第１６乃至２０番目のＴｈｒ−Ｇｌｕ−Ｐｈｅ−Ｔｒｐ−Ａｓｐで表される配列に基づき化学合成した５′−ＡＣＮＧＡＲＴＴＹＴＧＧＧＡ−３′で表される塩基配列のプローブ４をハイブリダイズさせ、顕著な会合を示した組換えＤＮＡを選択した。以上のようにして選択した組換えＤＮＡと形質転換体を、それぞれ、『ｐＢＱＴ１３』又は『ＢＱＴ１３』と命名した。
【００７４】
その後、この形質転換体ＢＱＴ１３をアンピシリンを含むＬ−ブロス培地で実施例１−２と同様に培養し、培養物より採取した菌体から組換えＤＮＡを溶出させ、精製し、分析したところ、組換えＤＮＡ ｐＢＱＴ１３は約７，２００残基対からなり、図１０に示す制限酵素地図で表される構造を有していた。図１０に示すように、酵素Ｑ３６をコードする２，３２５塩基対からなるＤＮＡは、制限酵素Ｘｍｎ Ｉによる切断部位付近の下流に位置していることが判明した。
【００７５】
【実施例３−３ 形質転換体ＢＱＴ１３による酵素の産生】
マルトース２．０％（ｗ／ｖ）、ペプトン０．５％（ｗ／ｖ）、酵母エキス０．１％（ｗ／ｖ）、燐酸水素二ナトリウム０．１％（ｗ／ｖ）、燐酸二水素カリウム０．１％（ｗ／ｖ）を含む液体培地をｐＨ７．０に調整し、アンピシリンを５０μｇ／ｍｌ加え、１２０℃で２０分間加熱滅菌し、冷却後、実施例３−２で得た形質転換体ＢＱＴ１３を植菌し、３７℃で２４時間回転振盪培養した。培養物を超音波処理して菌体を破砕し、遠心分離により不溶物を除去後、上清中の酵素活性を測定したところ、培養物１ｌ当たりに換算して、約２，４５０単位の酵素が産生していた。
【００７６】
別途、対照として、大腸菌ＸＬＩ−Ｂｌｕｅ株及びアルスロバクター・スピーシーズＱ３６をアンピシリン無含有の同じ組成の液体培地に植菌し、アルスロバクター・スピーシーズＱ３６の場合、培養温度を３０℃に設定した以外は上記と同様に培養・処理した。処理物の活性を測定したところ、アルスロバクター・スピーシーズＱ３６による酵素の産生は培養物１ｌ当たり約１，２００単位と、形質転換体ＢＱＴ１３と比較して有意に低いものであった。なお、宿主に使用した大腸菌ＸＬＩ−Ｂｌｕｅ株は、当該酵素を産生しなかった。
【００７７】
その後、形質転換体ＢＱＴ１３が産生した酵素を実験例１−１と同様に精製し、その性質・性状を調べたところ、ＳＤＳ−ポリアクリルアミドゲル電気泳動で分子量約７６，０００乃至８７，０００ダルトンを、また、等電点電気泳動で約３．６乃至４．６に等電点を示すなど、実験例２で得られた酵素Ｑ３６のものと同様の理化学的性質を有することが判明した。このことは、組換えＤＮＡ技術によっても当該酵素を製造でき、且つ、酵素の生産性も有意に向上することを示唆している。
【００７８】
【実施例４ アルスロバクター・スピーシーズＱ３６に由来する相補鎖ＤＮＡの調製とその塩基配列、アミノ酸配列の決定】
実施例３−２で得た組換えＤＮＡ ｐＢＱＴ１３を実施例２と同様に処理してテンプレートＤＮＡとし、これをプライマー１とともにアニーリング後、Ｔ７ ＤＮＡポリメラーゼを作用させてプライマー１を５′末端から３′末端に向かって伸長させ、相補鎖ＤＮＡを生成させた。実施例２と同様に、この相補鎖ＤＮＡにジデオキシ・チェーン・ターミネータ法を適用し、ラジオグラム上に分離したＤＮＡ断片を解析した結果、相補鎖ＤＮＡは配列表における配列番号６に示す３，０７３塩基対からなる塩基配列を含んでいることが判明した。この塩基配列から推定されるアミノ酸配列は配列表における配列番号６に併記したとおりであり、このアミノ酸配列と配列表における配列番号８、１１又は１２に示すＮ末端アミノ酸配列、部分アミノ酸配列を比較したところ、配列番号８に示すＮ末端アミノ酸配列は配列表における配列番号６における第１乃至２０番目の配列に、また、配列番号１１又は１２の部分アミノ酸配列は配列表における配列番号６における第６０６乃至６２５番目又は第１１０乃至１２９番目の配列に一致した。これは、酵素Ｑ３６が配列表における配列番号２に示すアミノ酸配列を有するものであり、アルスロバクター・スピーシーズＱ３６においては、酵素Ｑ３６が配列表における配列番号４に示す塩基配列のＤＮＡによりコードされていることを示している。
【００７９】
【発明の効果】
以上説明したように、この発明は、グルコース重合度３以上の還元性澱粉糖から末端にトレハロース構造を有する非還元性糖質を生成する、従来未知の全く新規な酵素の発見に基づくものである。この発明は、組換えＤＮＡ技術により斯かる酵素を大規模且つ効率的に生産する道を拓くものである。しかも、この発明による形質転換体が産生する酵素は、全アミノ酸配列までが明らかにされた酵素であり、食品等への配合使用を前提とするトレハロースや末端にトレハロース構造を有する非還元性糖質の製造に安心して使用し得るものである。
【００８０】
この発明は斯くも顕著な作用効果を奏する意義のある発明であり、斯界に貢献すること誠に多大な発明であると言える。
【００８１】
【配列表】

【００８２】

【００８３】

【００８４】

【００８５】

【００８６】

【００８７】

【００８８】

【００８９】

【００９０】

【００９１】

【００９２】

【図面の簡単な説明】
【図１】酵素Ｍ−１１の至適温度を示す図である。
【図２】酵素Ｑ３６の至適温度を示す図である。
【図３】酵素Ｍ−１１の至適ｐＨを示す図である。
【図４】酵素Ｑ３６の至適ｐＨを示す図である。
【図５】酵素Ｍ−１１の熱安定性を示す図である。
【図６】酵素Ｑ３６の熱安定性を示す図である。
【図７】酵素Ｍ−１１のｐＨ安定性を示す図である。
【図８】酵素Ｑ３６のｐＨ安定性を示す図である。
【図９】この発明による組換えＤＮＡであるｐＢＭＴ７の制限酵素地図である。なお、図中、太線表示部は、酵素Ｍ−１１をコードするＤＮＡを示す。
【図１０】この発明による組換えＤＮＡであるｐＢＱＴ１３の制限酵素地図である。なお、図中、太線表示部は、酵素Ｑ３６をコードするＤＮＡを示す。[0001]
[Industrial applications]
The present invention relates to a novel DNA encoding an enzyme for producing a non-reducing carbohydrate having a trehalose structure at a terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more, a recombinant DNA containing the DNA, and a transformant. Things.
[0002]
[Prior art]
Trehalose is a disaccharide in which two glucose molecules are bonded to each other with a reducing group, and is naturally present in trace amounts in bacteria, fungi, algae, insects and the like. Since trehalose does not have a reducing group in the molecule, it has the advantage that it does not cause a browning reaction even when heated in the presence of amino acids, and can sweeten foods without fear of coloration or deterioration. However, it is difficult to obtain a desired amount by the conventional production method, and it is hardly used for sweetening foods and drinks.
[0003]
Conventional production methods are broadly classified into a method using microbial cells and a method in which a complex enzyme system acts on saccharide. The former method involves growing microorganisms such as bacteria and yeast in a nutrient medium and collecting trehalose from the cells in the culture, as described in JP-A-50-154485. On the other hand, the latter method uses maltose as a substrate and acts with a complex enzyme system composed of maltose phosphorylase and trehalose phosphorylase as described in JP-A-58-216695. The resulting trehalose is taken out of the system. In the former method, although the growth of the microorganism itself is relatively easy, a series of complicated steps are required to collect trehalose from the cells, and the trehalose contained in the cells is 15% (w / w). There was a problem that it was negligible. In the latter method, although the separation of trehalose itself is relatively easy, the reaction itself is an equilibrium reaction by two kinds of enzymes, and the equilibrium is always inclined toward the glucose phosphate side. And increasing the yield of trehalose in principle.
[0004]
In view of such a situation, the present inventor has conducted an intensive search for an enzyme that produces a saccharide having a trehalose structure from starch sugar, and found that microorganisms such as Rhizobium sp. M-11 and Arthrobacter sp. It has been found that a completely novel enzyme, which is a conventionally unknown enzyme, that produces a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a degree of 3 or more. Before and after this finding, this non-reducing saccharide can be almost quantitatively converted to trehalose and glucose and / or maltooligosaccharide by another enzyme also produced by Rhizobium species M-11 or Arthrobacter species Q36. Was found to be hydrolyzed. By using these enzymes in combination, a desired amount of trehalose can be obtained relatively easily using starch as a raw material, and it is expected that the above problems relating to trehalose will be completely solved. However, neither Rhizobium species M-11 nor Arthrobacter species Q36 have sufficient production ability of the enzyme, and when trying to produce trehalose or a non-reducing saccharide having a trehalose structure at a terminal on a large scale, microorganisms are produced. There is a problem that a large amount of culture is required.
[0005]
On the other hand, recent advances in recombinant DNA technology have been remarkable. Nowadays, even for an enzyme whose entire amino acid sequence has not been elucidated, a gene encoding the enzyme has been isolated, and if its base sequence can be elucidated, a recombinant DNA containing the DNA encoding the enzyme has been produced. By culturing a transformant obtained by introducing it into a cell of a microorganism or animal or plant, a desired amount of enzyme can be obtained relatively easily. In view of such a situation, it is urgently necessary to identify genes encoding both enzymes and elucidate their base sequences.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a DNA encoding an enzyme that produces a non-reducing carbohydrate having a trehalose structure at a terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more.
[0007]
Another object of the present invention is to provide a recombinant DNA comprising the DNA and an autonomously replicable vector.
[0008]
Still another object of the present invention is to provide a transformant obtained by appropriately introducing the recombinant DNA into a host.
[0009]
[Means for Solving the Problems]
The present invention solves the first problem with a DNA encoding an enzyme that produces a non-reducing saccharide having a trehalose structure at a terminal from a reducing starch saccharide having a glucose polymerization degree of 3 or more.
[0010]
The second object of the present invention is to provide a DNA encoding an enzyme that produces a non-reducing saccharide having a trehalose structure at a terminal from a reducing starch sugar having a degree of glucose polymerization of 3 or more, and a vector capable of autonomously replicating. The problem is to be solved by using a replicable recombinant DNA.
[0011]
The third object of the present invention is to provide a DNA encoding an enzyme that produces a non-reducing carbohydrate having a trehalose structure at a terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more, and a vector capable of autonomous replication. This problem is solved by a transformant obtained by appropriately introducing a replicable recombinant DNA into a host.
[0012]
[Action]
The DNA of the present invention is inserted into an appropriate autonomously replicable vector to obtain a replicable recombinant DNA, and this recombinant DNA is used in a host that does not originally produce the enzyme but can be relatively easily propagated. By introducing the transformant, the production of the encoded enzyme is expressed.
[0013]
The recombinant DNA of the present invention, which does not naturally produce the enzyme, is transformed into a transformant by introducing it into a host that can be grown relatively easily. Express the production of the enzyme.
[0014]
The transformant of the present invention produces the enzyme when cultured.
[0015]
Hereinafter, the present invention will be described based on experimental examples, examples, and the like. The present invention relates to a method for producing a non-reducing saccharide having a trehalose structure at a terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. The present invention relates to a DNA encoding a novel enzyme. Such an enzyme can be obtained from a culture of Rhizobium species M-11 or Arthrobacter sp. Q36 (hereinafter referred to as "enzyme M-11" or "enzyme Q36", respectively), and the present inventors have made columns. The enzyme was isolated by a combination of various purification methods such as chromatography, and its properties and properties were examined. As a result, it was found that the enzyme was a polypeptide and had the following physicochemical properties.
(1) Action
A non-reducing saccharide having a trehalose structure at a terminal is produced from a reducing starch saccharide having a glucose polymerization degree of 3 or more.
(2) Molecular weight
Approximately 76,000 to 87,000 daltons (SDS-polyacrylamide gel electrophoresis)
(3) Isoelectric point
Approximately 3.6 to 4.6 (isoelectric focusing)
(4) Optimal temperature
Incubation at pH 7.0 for 60 minutes shows an optimum temperature around 35-40 ° C.
(5) Optimum pH
Incubation at 40 ° C. for 60 minutes shows an optimum pH around pH 6.4 to 7.2.
(6) Thermal stability
Incubation at pH 7.0 for 60 minutes is stable up to around 35-40 ° C.
(7) pH stability
When incubated at 25 ° C. for 16 hours, the pH is stable up to about 5.5 to 11.0.
[0016]
Enzymes having such physicochemical properties are not yet known and are considered to be novel substances. In addition, Rhizobium species M-11 was separated from the soil of Okayama city, Okayama prefecture, and from December 24, 1992, 1-3-1 Higashi, Tsukuba city, Ibaraki prefecture. Deposited with the National Institute of Advanced Industrial Science and Technology, the Patent Microorganisms Depositary under the deposit number "FERM BP-4130". On the other hand, Arthrobacter species Q36 was isolated from the soil of Soka City, Okayama Prefecture, and has been deposited with the center under the deposit number "FERM BP-4316" since June 3, 1993. By the same applicant JP-A-7-143876 ( Japanese Patent Application No. 5-349216 ) Discloses in detail the properties and properties of the enzyme, as well as the mycological properties of both microorganisms.
[0017]
The present inventor examined the partial amino acid sequence of the highly purified enzyme M-11, and as a result of intensively searching the chromosomal DNA of Rhizobium sp. M-11 using an oligonucleotide chemically synthesized based on the partial amino acid sequence as a probe, A DNA fragment consisting of 2,316 base pairs having the base sequence shown in SEQ ID NO: 3 in the sequence listing was obtained. When the base sequence was decoded, it was found that the enzyme M-11 was composed of 772 amino acids as shown in SEQ ID NO: 1 in the sequence listing.
[0018]
On the other hand, using the oligonucleotide chemically synthesized based on the partial amino acid sequence of the enzyme Q36 as a probe, the chromosomal DNA of Arthrobacter sp. Q36 was similarly searched, and it was found that 2,325 having the base sequence shown in SEQ ID NO: 4 in the sequence listing was obtained. A DNA fragment consisting of base pairs was obtained. Decoding this base sequence revealed that the enzyme Q36 was composed of 775 amino acids and had the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
[0019]
A series of steps leading to elucidation of the base sequence and amino acid sequence shown in SEQ ID NOs: 1 to 4 in the sequence listing are summarized as follows.
(1) The enzyme was isolated from the culture of the donor microorganism and highly purified. After partial hydrolysis of the purified enzyme with a protease, two types of peptide fragments were isolated from the hydrolyzate, and their amino acid sequences were determined.
(2) Separately, chromosomal DNA was separated from the cells of the donor microorganism, purified, and partially cut with a restriction enzyme to collect a DNA fragment consisting of about 3,000 to 7,000 base pairs. This DNA fragment was ligated with a DNA ligase to a plasmid vector that had been cut with a restriction enzyme in advance to prepare a recombinant DNA.
(3) A transformant is prepared by introducing a recombinant DNA into Escherichia coli, and a transformant containing a DNA encoding the enzyme is obtained by colony hybridization using an oligonucleotide chemically synthesized based on the partial amino acid sequence as a probe. Selected.
(4) Recombinant DNA is collected from the transformant, annealed together with the primer, and then the DNA polymerase is used to extend the primer. The obtained complementary DNA is analyzed by the dideoxy chain terminator method to determine the nucleotide sequence. Were determined. Then, the amino acid sequence deduced from the base sequence was compared with the partial amino acid sequence, and it was confirmed that the base sequence encoded the enzyme.
[0020]
The amino acid sequence encoded by the gene of the donor microorganism is as shown in SEQ ID NO: 1 or 2 in the Sequence Listing. The DNA of the present invention encodes the amino acid sequence as shown in SEQ ID NO: 1 or 2 in the Sequence Listing. Needless to say, those having an amino acid sequence homologous thereto are also included. That is, advances in recombinant DNA technology have made it possible in the art to relatively easily replace one or more of its constituent amino acids with another amino acid without substantially altering the action of the enzyme. . Even if the same DNA is used, depending on the host into which the DNA is introduced, the components and composition of the nutrient medium used for culturing the transformant containing the DNA, the culture temperature and pH, etc. Due to modification after expression, etc., the desired enzymatic action is maintained, but one or more amino acids near the N-terminal in the amino acid sequence of SEQ ID NO: 1 or 2 in the sequence listing are deleted, Mutants may be produced in which one or more amino acids are newly added at the N-terminus. In view of such a state of the art, the enzyme referred to in the present invention is not limited to an enzyme having the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing as it is, but one or more amino acids in the amino acid sequence. Is substituted or deleted or added to another amino acid as long as it forms a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. Shall be.
[0021]
Furthermore, in the art, one or more bases in DNA can be replaced by other bases without changing the amino acid sequence to be encoded due to the degeneracy of the genetic code. Thus, the DNA of the present invention is not limited to the DNA having the nucleotide sequence shown in SEQ ID NO: 3 or 4 as it is in the sequence listing, but also the enzyme having the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing or a homologous mutation thereof. As long as it encodes a body, it also includes one in which one or more bases are replaced with other bases based on the degeneracy of the genetic code.
[0022]
In addition, when the current recombinant DNA technology is used, generally, if a base sequence from the 5 'end is determined, a base sequence complementary thereto is uniquely determined. Therefore, the DNA of the present invention includes those having a base sequence complementary to any of the above base sequences. In order for the DNA of the present invention to actually express the production of the enzyme in a host, one or more bases in the base sequence encoding the enzyme or a homologous mutant thereof are appropriately substituted with another base. It goes without saying that it can be done.
[0023]
Although the DNA of the present invention is as described above, the DNA of the present invention does not matter whether it is naturally derived or artificially synthesized, as long as it has the sequence as described above. . Examples of natural sources include, for example, Rhizobium species M-11 (FERM BP-4130), Arthrobacter species Q36 (FERM BP-4316), Brevibacterium herovolum (ATCC 11822), and Flavobacterium aquatile (IFO3772). ), Micrococcus luteus (IFO 3064), Micrococcus roseus (ATCC 186), Curtobacterium citreum (IFO 15231), Mycobacterium smegmatis (ATCC 19420) and Rhizobium spp. Genus, Brevibacterium, Flavobacterium, Micrococcus, Curtobacterium, Mycobacterium, Terabactor Goods and the like, from the cells of these microorganisms gene comprising DNA of the invention is obtained. That is, such a microorganism is inoculated into a nutrient medium, cultured under aerobic conditions for about 1 to 3 days, and then the cells are collected from the culture and subjected to lysozyme, β-glucanase or other cell wall lysing enzymes or ultrasonic waves. By performing the treatment, the gene containing the DNA is eluted out of the cells. At this time, a protein hydrolase such as protease may be used in combination with the cell wall lysing enzyme, or a surfactant such as SDS may be coexistent or freeze-thawed when the cells are subjected to ultrasonic treatment. A target DNA can be obtained by applying a general method in the art such as phenol extraction, alcohol precipitation, centrifugation, protease treatment, ribonuclease treatment and the like to the thus-treated product.
[0024]
On the other hand, in order to artificially synthesize the DNA of the present invention, for example, chemical synthesis is performed based on the nucleotide sequence shown in SEQ ID NO: 3 or 4 in the sequence listing, or the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing is obtained. The encoding DNA is inserted into an appropriate vector capable of autonomous replication to obtain a recombinant DNA, and a transformant obtained by introducing this into an appropriate host is cultured, cells are separated from the culture, and the cells are isolated from the cells. A plasmid containing DNA may be collected.
[0025]
By the way, the present invention also relates to a replicable recombinant DNA which expresses the production of the enzyme when introduced into a cell of a microorganism or animal or plant which does not originally produce the enzyme but can be relatively easily proliferated. . Such a recombinant DNA usually comprises a DNA as described above and a vector capable of autonomous replication, and if the DNA is available, it can be prepared relatively easily by ordinary recombinant DNA techniques. Examples of such vectors include plasmid vectors such as pBR322, pUC18, Bluescript II SK (+), pUB110, pTZ4, pC194, pHV14, TRp7, YEp7, pBS7, and λgt · λC, λgt · λB, ρ11, φ1, φ105. Among them, pBR322, pUC18, Bluescript II SK (+), λgt.λC and λgt.λB are preferable for expressing the DNA of the present invention in Escherichia coli. For expression, pUB110, pTZ4, pC194, ρ11, φ1, and φ105 are suitable. pHV14, TRp7, YEp7 and pBS7 are useful when growing recombinant DNA in more than one host.
[0026]
In order to insert the DNA of the present invention into such a vector, a method generally used in the art is employed. Specifically, first, the gene containing the DNA of the present invention and an autonomously replicable vector are cut with a restriction enzyme and / or ultrasonic waves, and then the generated DNA fragment and the vector fragment are ligated. Restriction enzymes that specifically act on nucleotides for the cleavage of genes and vectors, especially type II restriction enzymes, specifically Sau 3AI, Eco RI, Hind III, Bam HI, Sal I, Xba I, Sac I, Pst Use of I or the like facilitates ligation of the DNA fragment and the vector fragment. In order to ligate the DNA fragment and the vector fragment, they may be annealed, if necessary, and then subjected to DNA ligase in vivo or in vitro. The recombinant DNA thus obtained can be replicated indefinitely by appropriately introducing it into a host to form a transformant and culturing the transformant.
[0027]
The recombinant DNA according to the present invention can be introduced into an appropriate host microorganism such as Escherichia coli, Bacillus subtilis, actinomycetes, and yeast. When the host is Escherichia coli, the host may be cultured in the presence of recombinant DNA and calcium ions. On the other hand, when the host is Bacillus subtilis, the competent cell method or the protoplast method may be applied. To clone a transformant, a colony hybridization method is applied, or the transformant is cultured in a nutrient medium containing a reducing starch sugar having a glucose polymerization degree of 3 or more, and a non-reducing sugar having a trehalose structure at a terminal from the starch sugar. Whatever produces quality can be selected.
[0028]
When the transformant thus obtained is cultured in a nutrient medium, the enzyme is produced inside and outside the cells. As the nutrient medium, a carbon source, a nitrogen source, minerals, and, if necessary, a general liquid medium supplemented with trace nutrients such as amino acids and vitamins are used, and as individual carbon sources, for example, Carbohydrates such as starch, starch hydrolysate, glucose, fructose, and sucrose; and nitrogen sources such as ammonia or ammonium salts, urea, nitrate, peptone, yeast extract, defatted soybean, corn steep liquor, and meat. Examples include nitrogen-containing inorganic or organic substances such as extracts. When the transformant is inoculated into the nutrient medium and cultured for about 1 to 6 days under aerobic conditions such as aeration and stirring while maintaining the nutrient medium at a temperature of 25 to 65 ° C. and a pH of 2 to 8, the enzyme Is obtained. This culture can be used as it is as an enzymatic agent, but usually, prior to use, if necessary, disrupt the cells with ultrasonic waves or cell wall lysing enzymes, and then filter or centrifuge the cells to remove the enzymes. It is separated from the crushed cells and purified. For the purification, a general method for purifying an enzyme can be employed.For example, concentration, salting out, dialysis, fractional precipitation, gel filtration chromatography, ion exchange chromatography One or a combination of two or more of chromatography, hydrophobic chromatography, affinity chromatography, gel electrophoresis, and isoelectric focusing may be applied as appropriate.
[0029]
As described above, the enzyme has a remarkable action not found in the conventional enzyme, that is, non-reducing saccharide having a trehalose structure at a terminal is produced from reducing starch sugar having a glucose polymerization degree of 3 or more. Therefore, the enzyme produced by the transformant of the present invention is maltotriose, maltotetraose, maltopentaose, maltohexaose obtained by treating starch or starchy substances such as amylopectin and amylose with an acid and / or amylase. And the like, by acting on a starch hydrolyzate containing a series of maltooligosaccharides having a glucose polymerization degree of 3 or more, α-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose having a trehalose structure at the terminal , And corresponding non-reducing saccharides such as α-maltotetraosyltrehalose can be efficiently produced with good yield. And these non-reducing carbohydrates JP-A-7-213283 ( Japanese Patent Application No. 5-340343 ) Trehalose is produced almost quantitatively by the action of the trehalose-releasing enzyme disclosed in US Pat.
[0030]
Next, a series of experiments performed to elucidate the physicochemical properties of an enzyme for producing a non-reducing saccharide having a trehalose structure at the terminal from a reducing starch saccharide having a glucose polymerization degree of 3 or more will be described.
[0031]
[Experimental example 1 Preparation of purified enzyme]
[0032]
Experimental Example 1-1 Purification of Enzyme M-11
In a 500 ml Erlenmeyer flask, maltose 2.0% (w / v), peptone 0.5% (w / v), yeast extract 0.1% (w / v), disodium hydrogen phosphate 0.1% (w / v) 100 ml of a liquid medium (pH 7.0) containing v) and potassium dihydrogen phosphate 0.1% (w / v) was taken, and sterilized by autoclaving at 120 ° C. for 20 minutes. After cooling, Rhizobium species M-11 was inoculated into the liquid medium in the Erlenmeyer flask, and seed-cultured at 27 ° C. for 24 hours under rotary shaking. Separately, 20 l of a liquid medium having the same composition as above is placed in a 30-liter jar fermenter, and after sterilization, 1% (v / v) of the seed culture obtained above is inoculated, while maintaining the liquid medium at pH 6 to 8, Incubation with aeration and stirring was performed at 30 ° C. for 24 hours.
[0033]
Next, about 18 l of the culture obtained above was placed in an ultrahigh-pressure cell disruption apparatus, and after disrupting the cells, ammonium sulfate was added to about 16 l of the supernatant collected by centrifugation so that the concentration became 20% saturated. After standing for 1 hour, the precipitate was removed by centrifugation. Ammonium sulfate was added to the obtained supernatant so as to be 60% saturated, and the mixture was allowed to stand at 4 ° C. for 24 hours. The precipitate was collected by centrifugation and dissolved in a minimum amount of 10 mM phosphate buffer (pH 7.0). After dialysis against 10 mM phosphate buffer (pH 7.0) for 24 hours, insoluble materials were removed by centrifugation. The obtained supernatant was loaded on a column for ion exchange chromatography "DEAE-Toyopearl" manufactured by Tosoh, which had been equilibrated with 10 mM phosphate buffer (pH 7.0) in advance, and sodium chloride rising from 0 M to 0.5 M was used. 10 mM phosphate buffer (pH 7.0) was passed through the column under a concentration gradient of 1. A fraction containing the enzyme was collected from the eluate, dialyzed against 50 mM phosphate buffer (pH 7.0) containing 2 M ammonium sulfate for 10 hours, and centrifuged to remove insolubles. Thereafter, the supernatant was loaded on a column “butyl toyopearl” for hydrophobic chromatography manufactured by Tosoh, which had been equilibrated with a 50 mM phosphate buffer (pH 7.0) containing 2 M ammonium sulfate in advance, and ammonium sulfate was reduced from 2 M to 0 M. Under a concentration gradient, 50 mM phosphate buffer (pH 7.0) was passed through the column. A fraction containing an enzyme was collected from the eluate, loaded onto a column for gel filtration column chromatography “Toyopearl HW-55” manufactured by Tosoh, which had been equilibrated with 50 mM phosphate buffer (pH 7.0), and loaded onto the column. After passing through a 50 mM phosphate buffer (pH 7.0), a fraction containing the enzyme was collected from the eluate. The specific activity of the thus purified enzyme M-11 was about 195 units / mg protein, and the yield was about 220 units per liter of culture.
[0034]
Throughout the present invention, the activity of an enzyme is represented by an activity value (unit) measured by the following method. That is, 4 ml of 50 mM phosphate buffer (pH 7.0) containing 1.25% (w / v) maltopentaose, 1 ml of the enzyme solution was added thereto, and the mixture was incubated at 40 ° C. for 60 minutes to react. The reaction is heated at 100 ° C. for 10 minutes to stop the reaction. After diluting the reaction solution 10 times with distilled water, the reducing power is measured by the Somogi-Nelson method. One unit of the enzyme is defined as the amount of the enzyme that reduces the reducing power corresponding to 1 μmol of maltopentaose per minute under the above conditions.
[0035]
[Experimental example 1-2 Purification of enzyme Q36]
When Arthrobacter sp. Q36 was cultured and the culture was treated in the same manner as in Experimental Example 1-1, the purified enzyme Q36 having a specific activity of about 200 units / mg protein was obtained at a yield of about 295 units per liter of culture. Obtained.
[0036]
[Experimental example 2 Physicochemical properties of enzyme]
In this experimental example, using the purified enzyme obtained in Experimental Example 1 as a sample, the physicochemical properties of the enzyme are examined.
[0037]
[Experimental example 2-1 Action]
It was obtained in Experimental Example 1 in a 50 mM phosphate buffer (pH 7.0) containing 20% (w / v) of glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose or maltoheptaose as a substrate. Purified enzyme M-11 or purified enzyme Q36 was added at 2 units per 1 g of the substrate, and reacted at 40 ° C. for 48 hours. After desalting the reaction product by a conventional method, the reaction product was loaded on a high-performance liquid chromatography column “WB-T-330” manufactured by Wako Pure Chemical Industries, and the sugar concentration of the eluate was measured using a differential refractometer “RI-8012 type” manufactured by Tosoh. While monitoring, carbohydrate contained in the reaction product was separated by passing distilled water through the column at a flow rate of 0.5 ml / min at room temperature. Tables 1 and 2 show the sugar compositions when the enzymes M-11 and Q36 were added, respectively. The sugars P1 to P5 in the table are obtained by naming the sugars produced by the reaction in ascending order of glucose polymerization degree.
[0038]
[Table 1]

[0039]
[Table 2]

[0040]
As is clear from the results of Tables 1 and 2, the enzymes M-11 and Q36 have a degree of polymerization of glucose of 3 or more, such as maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. Reducing starch sugars produce new carbohydrates, but do not produce new carbohydrates from glucose or maltose with a degree of glucose polymerization of less than 3. The carbohydrates produced by the reaction were only carbohydrates P1 to P5, respectively, and the content of carbohydrates P2 to P5 was remarkably high at 85% or more per solid.
[0041]
Next, in order to separate the carbohydrates P1 to P5, a strongly acidic cation exchange resin "XT-1016 (Na ⁺ Was packed in three stainless steel columns with a jacket having an inner diameter of 2.0 cm and a length of 1 m, and these columns were connected in series. Then, while maintaining the temperature in the column at 55 ° C, the reactants containing any of the carbohydrates P1 to P5 were separately loaded onto the column, and then distilled water at 55 ° C was passed through the column at a flow rate of SV 0.13. Liquid. The sugar composition of the eluate was examined, and a fraction containing 97% or more of any of the carbohydrates P1 to P5 in solid content was collected and powdered by vacuum drying. When the reducing power of the carbohydrates P1 to P5 thus purified was examined by the Somogi-Nelson method, no substantial reducing power was observed for any of the carbohydrates.
[0042]
Further, in order to identify carbohydrates P1 to P5, 50 mg of any of these carbohydrates was taken, dissolved in 1 ml of 50 mM acetate buffer (pH 4.5), 1 unit of glucoamylase was added, and the mixture was incubated at 40 ° C. for 6 hours. . When the sugar compositions of the reactants shown in Tables 1 and 2 were analyzed by high performance liquid chromatography, glucose and trehalose were detected from all the reactants as shown in Tables 3 and 4, respectively. Similarly, when β-amylase was allowed to act on carbohydrates P1 to P5, carbohydrates P1 and P2 were not affected by β-amylase, whereas carbohydrate P3 contained one molecule of maltose and carbohydrate. Carbohydrate P4 provided one molecule of maltose and carbohydrate P2, and carbohydrate P5 provided two molecules of maltose and carbohydrate P1.
[0043]
[Table 3]

[0044]
[Table 4]

[0045]
The results in Tables 3 and 4 strongly suggest that carbohydrates P1 to P5 are composed of one molecule of trehalose and one to five molecules of glucose. Further, glucoamylase specifically cleaves at α-1,4 bond and α-1,6 bond in maltooligosaccharide, and β-amylase converts α-1,4 bond in maltooligosaccharide in maltose units from the terminal. From the cleavage, it is presumed that the carbohydrates P1 to P5 have a structure in which one trehalose residue is bonded to the terminal of glucose or a maltooligosaccharide having a glucose polymerization degree of 2 to 5.
[0046]
Comprehensively judging the above results, the carbohydrates P1 to P5 were respectively α-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose, α-maltotetraosyltrehalose or α-maltopentaoate. It was identified as siltrehalose, which supports that the enzyme has an action of producing a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more.
[0047]
[Experimental example 2-2 Molecular weight]
Purified enzyme was subjected to SDS-polyacrylamide gel electrophoresis according to the method reported by U.K.Remli in "Nature", Vol. 227, pp. 680-685 (1970). For the enzyme Q36, a single band was observed at a position corresponding to a molecular weight of about 76,000 to 87,000 daltons. The molecular weight markers at this time were myosin (200,000 dalton), β-galactosidase (116,250 dalton), phosphorylase B (97,400 dalton), serum albumin (66,200 dalton), and ovalbumin ( 45,000 daltons).
[0048]
[Experimental example 2-3 isoelectric point]
When measured by isoelectric focusing, both the enzyme M-11 and the enzyme Q36 showed an isoelectric point of about 3.6 to 4.6.
[0049]
[Experimental example 2-4 Optimum temperature]
According to a conventional method, the test was performed under the condition of incubating for 60 minutes in a 50 mM phosphate buffer (pH 7.0). As shown in FIG. 1 or FIG. 2, both the enzyme M-11 and the enzyme Q36 were at around 35 to 40 ° C. The optimum temperature was indicated.
[0050]
[Experimental example 2-5 Optimum pH]
According to a conventional method, the test was performed under the conditions of incubation at 40 ° C. for 60 minutes in 50 mM acetate buffer, phosphate buffer or sodium carbonate-sodium bicarbonate buffer having different pHs. As shown in FIG. 3 or FIG. Both the enzyme M-11 and the enzyme Q36 showed the optimum pH around pH 6.4 to 7.2.
[0051]
[Experimental example 2-6 Thermal stability]
According to a conventional method, the test was performed under the condition of incubating for 60 minutes in a 50 mM phosphate buffer (pH 7.0). As shown in FIG. 5 or FIG. 6, both the enzyme M-11 and the enzyme Q36 were heated to around 35 to 40 ° C. It was stable.
[0052]
[Experimental example 2-7 pH stability]
According to a conventional method, the test was performed under the conditions of incubation at 25 ° C. for 16 hours in 50 mM acetate buffer, phosphate buffer or sodium carbonate-sodium bicarbonate buffer having different pH, and as shown in FIG. 7 or FIG. Both the enzyme M-11 and the enzyme Q36 were stable from pH 5.5 to around 11.0.
[0053]
[Experimental example 2-8 N-terminal amino acid sequence]
Analysis by a conventional method using a gas-phase protein sequencer “470A” manufactured by Applied Biosystems revealed that the enzyme M-11 had the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing at the N-terminus. It has been found.
[0054]
Similar analysis revealed that the enzyme Q36 had an amino acid sequence represented by SEQ ID NO: 8 in the sequence listing at the N-terminus.
[0055]
[Experimental example 2-9 Partial amino acid sequence]
An appropriate amount of the purified enzyme M-11 obtained in Experimental Example 1-1 was taken, dialyzed against 10 mM Tris-HCl buffer (pH 9.0) at 4 ° C for 18 hours, and then 10 mM Tris-HCl buffer (pH 9.0). Was added to adjust the enzyme concentration to about 1 mg / ml. About 1 ml of this solution was added, 10 μg of lysyl endopeptidase was added, and the mixture was incubated at 30 ° C. for 22 hours to partially hydrolyze the enzyme. The column “Capsule Pack C18” for reverse phase high performance liquid chromatography manufactured by Shiseido, in which the hydrolyzate was previously equilibrated with 0.1% (v / v) trifluoroacetic acid containing 16% (v / v) aqueous acetonitrile. Under a gradient of acetonitrile from 16% (v / v) to 64% (v / v) with 0.1% (v / v) trifluoroacetic acid at 0.9 ml / At a flow rate of 1 min. Then, fractions containing peptide fragments (hereinafter, referred to as “peptide fragment A” or “peptide fragment B,” respectively) eluted about 28 minutes or about 40 minutes after the start of the passage are separately collected and dried in vacuo. Afterwards, it was dissolved in 0.1% (v / v) trifluoroacetic acid containing 50% (v / v) aqueous acetonitrile. Thereafter, analysis was performed in the same manner as in Experimental Example 2-8, and it was found that peptide fragments A and B had the amino acid sequences shown in SEQ ID NOS: 9 and 10 in the sequence listing.
[0056]
Separately, the purified enzyme Q36 obtained in Experimental Example 1-2 is partially hydrolyzed in the same manner as described above, and equilibrated with 0.1% (v / v) trifluoroacetic acid containing 24% (v / v) aqueous acetonitrile in advance. Of aqueous acetonitrile, which is loaded on the column "Micro Bonder Pack C18" for reversed phase high performance liquid chromatography manufactured by Nippon Millipore Limited and increased from 24% (v / v) to 44% (v / v) Under the gradient, 0.1% (v / v) trifluoroacetic acid was passed through the column at a flow rate of 0.9 ml / min. Then, a fraction containing the peptide fragment (hereinafter, referred to as “peptide fragment C” or “peptide fragment D”) eluted about 22 minutes or about 40 minutes after the start of the liquid passage, is collected, dried in vacuo, Dissolved in 0.1% (v / v) trifluoroacetic acid containing 50% (v / v) aqueous acetonitrile. Thereafter, the same analysis as above revealed that peptide fragments C and D had the amino acid sequences shown in SEQ ID NOS: 11 and 12 in the sequence listing.
[0057]
The physicochemical properties of the enzyme M-11 and the enzyme Q36 are as described above. In the following Examples, based on these physicochemical properties, a replicable recombinant DNA and a transformant according to the present invention were prepared, The DNA encoding the enzyme contained therein is collected, and its base sequence and amino acid sequence are determined. The techniques used in these examples are known in the art. For example, J. Sambrook et al., "Molecular Cloning A Laboratory Manual", 2nd edition, Cold Spring Harbor, 1989. It is also described in detail in the laboratory press.
[0058]
Example 1 Preparation of Recombinant DNA Containing DNA Derived from Rhizobium sp. M-11 and Transformant
[0059]
[Example 1-1 Preparation of chromosomal DNA]
Rhizobium species M-11 was inoculated in a Bacto-nutrient broth medium (pH 7.0), and cultured at 27 ° C. for 24 hours with rotary shaking. Cells were separated from the culture by centrifugation, suspended in TES buffer (pH 8.0), lysozyme was added at 0.05% (w / v), and then incubated at 37 ° C for 30 minutes. After the processed product is frozen at -80 ° C for 1 hour, a TSS buffer (pH 9.0) is added, the mixture is heated to 60 ° C, a TES buffer / phenol mixed solution is added, and after cooling with ice, the supernatant is collected by centrifugation. did. Two volumes of cold ethanol was added to the supernatant, and the precipitated crude chromosomal DNA was collected and dissolved in SSC buffer (pH 7.1). Then, 7.5 μg or 125 μg of ribonuclease and protease were added thereto, and 1 hour at 37 ° C. Incubate for hours to react. Thereafter, a mixed solution of chloroform / isoamyl alcohol was added to the reaction product to extract chromosomal DNA, cold ethanol was added, and a precipitate containing the generated chromosomal DNA was collected. The purified chromosomal DNA thus obtained was dissolved in SSC buffer (pH 7.1) to a concentration of about 1 mg / ml, and the solution was frozen at -80 ° C.
[0060]
Example 1-2 Preparation of Recombinant DNA pBMT7 and Transformant BMT7 About 1 ml of the purified chromosomal DNA solution obtained in Example 1-1 was added, and about 35 units of the restriction enzyme Sau 3AI was added thereto. After allowing a reaction for about 20 minutes to partially cut the chromosomal DNA, a DNA fragment consisting of about 3,000 to 7,000 base pairs was collected by sucrose density gradient ultracentrifugation. Separately, 1 μg of the plasmid vector Bluescript II SK (+) was taken and completely digested with the restriction enzyme BamHI by a conventional method. Then, 10 μg of the DNA fragment obtained above and 2 units of T4 DNA ligase were added, and the mixture was added at 4 ° C. The DNA fragment was ligated to the vector fragment by standing overnight. Then, 30 μl of a competent cell “Epicurian Coli XLI-Blue” manufactured by Toyobo Co., Ltd. was added to the obtained recombinant DNA, the mixture was allowed to stand under ice-cooling for 30 minutes, heated to 42 ° C., and added with SOC broth to 37 ° C. For 1 hour to introduce the recombinant DNA into E. coli.
[0061]
Next, the transformant obtained above was inoculated on an agar plate medium (pH 7.0) containing 50 μg / ml of 5-bromo-4-chloro-3-indolyl-β-galactoside, and cultured at 37 ° C. for 18 hours. Thereafter, a nylon membrane was placed on the medium, and about 4,400 colonies formed on the medium were fixed on the nylon membrane. Separately, based on the sequence represented by the 17th to 21st positions of Pro-Glu-Trp-Glu-Lys in the amino acid sequence represented by SEQ ID NO: 9 in the sequence listing, it is represented by 5'-CCNGARTGGGGARAA-3 '. Chemical synthesis of probe 1 with base sequence ³² After labeling with P, hybridization was carried out to colonies of the transformants fixed on the nylon membrane, and nine types of transformants in which remarkable association was recognized were selected.
[0062]
Recombinant DNA was collected from these nine transformants by a conventional method, and the sequence represented by Thr-Glu-Phe-Trp-Asp at the 16th to 20th amino acids in the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing Probe 2 having a nucleotide sequence represented by 5'-ACNGARTTYTGGGGA-3 'chemically synthesized based on E.M.S.Zern, Journal of Molecular Biology, Vol. 98, pp. 503-517 (1975) ), And a recombinant DNA showing a remarkable association with probe 2 was selected. The recombinant DNA and transformant selected as described above were named "pBMT7" or "BMT7", respectively.
[0063]
The transformant BMT7 obtained above was inoculated into an L-broth medium (pH 7.0) containing 100 μg / ml of ampicillin, and cultured at 37 ° C. for 24 hours with rotation and shaking. After completion of the culture, the cells were collected from the culture by centrifugation, and the recombinant DNA was eluted out of the cells by a general alkali method. The treated product was purified by a conventional method and analyzed. As a result, the recombinant DNA pBMT7 was composed of about 9,300 base pairs and had a structure represented by the restriction enzyme map shown in FIG. As shown in FIG. 9, it was found that the DNA consisting of 2,316 base pairs encoding the enzyme M-11 was located downstream near the site of cleavage by the restriction enzyme PstI.
[0064]
Example 1-3 Production of Enzyme by Transformant BMT7
Maltose 2.0% (w / v), Peptone 0.5% (w / v), Yeast extract 0.1% (w / v), Disodium hydrogen phosphate 0.1% (w / v), Diphosphate A liquid medium containing 0.1% (w / v) of potassium hydrogen was adjusted to pH 7.0, 50 μg / ml of ampicillin was added, and the mixture was sterilized by heating at 120 ° C. for 20 minutes, cooled, and then obtained in Example 1-2. The transformant BMT7 was inoculated and cultivated with rotation and shaking at 37 ° C. for 24 hours. The culture was sonicated to disrupt the cells, and the insoluble matter was removed by centrifugation. After measuring the enzyme activity in the supernatant, the enzyme activity was determined to be about 3,000 units per liter of culture. Was produced.
[0065]
Separately, as a control, Escherichia coli XLI-Blue strain and Rhizobium sp. M-11 were inoculated in the same liquid medium containing no ampicillin, and in the case of Rhizobium sp. M-11, the culture temperature was set to 30 ° C. Culture and treatment were carried out in the same manner as described above. When the activity of the treated product was measured, the production of the enzyme by Rhizobium sp. M-11 was about 1,500 units per liter of the culture, which was significantly lower than that of the transformant BMT7. The E. coli XLI-Blue strain used as the host did not produce the enzyme at all.
[0066]
Thereafter, the enzyme produced by the transformant BMT7 was purified in the same manner as in Experimental Example 1-1, and its properties and properties were examined. The molecular weight of the enzyme was about 76,000 to 87,000 daltons by SDS-polyacrylamide gel electrophoresis. Has the same physicochemical properties as that of the enzyme M-11 obtained in Experimental Example 2, such as showing an isoelectric point of about 3.6 to 4.6 by isoelectric focusing. did. This suggests that the enzyme can be produced by recombinant DNA technology, and that the productivity of the enzyme is also significantly improved.
[0067]
Example 2 Preparation of DNA of complementary strand derived from Rhizobium species M-11 and determination of its base sequence and amino acid sequence
The recombinant DNA pBMT7 obtained in Example 1-2 was digested with various restriction enzymes according to a conventional method, and subcloned into Bluescript II SK (+) to obtain a DNA for nucleotide sequence determination. 2 μg of each of these DNAs for nucleotide sequence determination was denatured by adding a 2M aqueous sodium hydroxide solution, and then an appropriate amount of cold ethanol was added. A precipitate containing the generated template DNA was collected and dried in vacuo. Primer 1 having a base sequence represented by 5′-GTAAAACGACGGCCAGT-3 ′ chemically synthesized with the template DNA was added at 50 pmol / ml and a 40 mM Tris-HCl buffer (pH 7.5) containing 20 mM magnesium chloride and 50 mM sodium chloride. After adding 10 µl and incubating at 65 ° C for 2 minutes for annealing, 2 µl of an aqueous solution containing 7.5 µM each of dATP, dGTP and dTTP was added, and [α- ³² P] dCTP (2 mCi / ml), 0.5 μl, 0.1 M dithiothreitol, 1 μl, 1.5 unit / ml T7 DNA polymerase 2 μl, and incubate at 25 ° C. for 5 minutes to primer 1 The DNA was extended from the 5 'end toward the 3' end to generate complementary DNA.
[0068]
Next, the reaction product containing the complementary strand DNA obtained above was divided into four equal parts, and 2.5 μl of a 50 mM sodium chloride aqueous solution containing 8 μM of either ddATP, ddCTP, ddGTP or ddTTP and 80 μM dNTP was added thereto, and 37 After incubation at 5 ° C. for 5 minutes, a 95% (v / v) aqueous solution containing 20 mM EDTA, 0.05% (w / v) bromophenol blue and 0.05% (w / v) xylene cyanol was used. The reaction was stopped by adding 4 μl of a formamide solution. The reaction product was heated in a boiling water bath for 3 minutes, then placed on a 6% (w / v) polyacrylamide gel, and subjected to electrophoresis while applying a constant voltage of about 2,000 V to separate DNA fragments. After the gel was fixed and dried, autoradiography was performed.
[0069]
As a result of analyzing the DNA fragment separated on the radiogram, it was found that the complementary strand DNA contained a base sequence consisting of 2,936 base pairs shown in SEQ ID NO: 5 in the sequence listing. The amino acid sequence deduced from this nucleotide sequence is as described in SEQ ID NO: 5 in the Sequence Listing, and the N-terminal amino acid sequence and part of the enzyme M-11 shown in SEQ ID NO: 7, 9 or 10 in the Sequence Listing When the amino acid sequences were compared, the N-terminal amino acid sequence of SEQ ID NO: 7 was the first to twentieth sequences of SEQ ID NO: 5 in the sequence listing, and the partial amino acid sequence of SEQ ID NO: 9 or 10 was the sequence of SEQ ID NO: 5 in the sequence listing. In the 486th to 506th sequences or the 606th to 626th sequences. This means that the enzyme M-11 has the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and in Rhizobium species M-11, the enzyme M-11 has the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing. Indicates that it is coded.
[0070]
Example 3 Preparation of Recombinant DNA Containing DNA Derived from Arthrobacter sp. Q36 and Transformant
[0071]
Example 3-1 Preparation of chromosomal DNA
Chromosomal DNA was separated and purified from Arthrobacter sp. Q36 in the same manner as in Example 1-1, dissolved in SSC buffer (pH 7.1) to a concentration of about 1 mg / ml, and frozen at -80 ° C. did.
[0072]
Example 3-2 Preparation of recombinant DNA pBQT13 and transformant BQT13
After the purified chromosomal DNA solution obtained in Example 3-1 was partially cut in the same manner as in Example 1-2, a DNA fragment consisting of about 3,000 to 6,000 base pairs was collected by sucrose density gradient ultracentrifugation. . Thereafter, using T4 DNA ligase, this DNA fragment was ligated to the digest of the vector Bluescript II SK (+) digested with the restriction enzyme Bam HI in the same manner as in Example 1-2, and the obtained recombinant DNA was subjected to Escherichia coli XLI- It was introduced into the Blue strain. The obtained transformant was cultured on an agar plate medium containing 5-bromo-4-chloro-3-indolyl-β-galactoside in the same manner as in Example 1-2. While immobilized on the membrane, 5′-TTYGAYGTNGAYTGGGGA-3 ′ based on the sequence represented by Phe-Asp-Val-Asp-Trp-Asp at positions 11 to 16 in the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing. Probe 3 having the nucleotide sequence represented was chemically synthesized and ³² After labeling with P, hybridization was performed with colonies of the transformants fixed on the nylon membrane, and eight types of transformants in which remarkable association was recognized were selected.
[0073]
In the same manner as in Example 1-2, recombinant DNAs were collected from these eight transformants, and Thr-Glu-Phe-Trp- at the 16th to 20th amino acids in the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing. Probe 4 having a nucleotide sequence represented by 5'-ACNGARTTYTGGGA-3 'chemically synthesized based on the sequence represented by Asp was hybridized, and a recombinant DNA showing a remarkable association was selected. The recombinant DNA and transformant selected as described above were named "pBQT13" or "BQT13", respectively.
[0074]
Thereafter, the transformant BQT13 was cultured in an L-broth medium containing ampicillin in the same manner as in Example 1-2. Recombinant DNA was eluted from the cells collected from the culture, purified, and analyzed. The recombinant DNA pBQT13 consisted of about 7,200 residue pairs and had a structure represented by the restriction map shown in FIG. As shown in FIG. 10, it was found that the DNA consisting of 2,325 base pairs encoding the enzyme Q36 was located downstream near the site of cleavage by the restriction enzyme XmnI.
[0075]
[Example 3-3 Production of enzyme by transformant BQT13]
Maltose 2.0% (w / v), Peptone 0.5% (w / v), Yeast extract 0.1% (w / v), Disodium hydrogen phosphate 0.1% (w / v), Diphosphate A liquid medium containing 0.1% (w / v) of potassium hydrogen was adjusted to pH 7.0, 50 μg / ml of ampicillin was added, and the mixture was sterilized by heating at 120 ° C. for 20 minutes, cooled, and then obtained in Example 3-2. The transformant BQT13 was inoculated and cultured at 37 ° C. for 24 hours with rotation and shaking. The culture was sonicated to disrupt the cells, and the insoluble matter was removed by centrifugation. After measuring the enzyme activity in the supernatant, the enzyme activity was determined to be about 2,450 units per liter of culture. Was produced.
[0076]
Separately, as a control, Escherichia coli XLI-Blue strain and Arthrobacter sp. Q36 were inoculated into a liquid medium having the same composition without ampicillin, and in the case of Arthrobacter sp. Q36, the culture temperature was set to 30 ° C. Was cultured and treated in the same manner as described above. When the activity of the treated product was measured, the production of the enzyme by Arthrobacter sp. Q36 was about 1,200 units per liter of the culture, which was significantly lower than that of the transformant BQT13. The E. coli XLI-Blue strain used as the host did not produce the enzyme.
[0077]
Thereafter, the enzyme produced by the transformant BQT13 was purified in the same manner as in Experimental Example 1-1, and its properties and properties were examined. By SDS-polyacrylamide gel electrophoresis, a molecular weight of about 76,000 to 87,000 daltons was obtained. Further, it was found that the enzyme Q36 had the same physicochemical properties as that of the enzyme Q36 obtained in Experimental Example 2, such as showing an isoelectric point of about 3.6 to 4.6 in isoelectric focusing. This suggests that the enzyme can be produced by recombinant DNA technology, and that the productivity of the enzyme is also significantly improved.
[0078]
Example 4 Preparation of DNA of Complementary Strand Derived from Arthrobacter sp. Q36 and Determination of Its Base Sequence and Amino Acid Sequence
The recombinant DNA pBQT13 obtained in Example 3-2 was treated in the same manner as in Example 2 to obtain a template DNA, which was annealed together with the primer 1 and then reacted with T7 DNA polymerase to place the primer 1 3 ′ from the 5 ′ end. Extension toward the ends produced complementary DNA strands. As in Example 2, the dideoxy chain terminator method was applied to this complementary strand DNA, and the DNA fragment separated on the radiogram was analyzed. As a result, the complementary strand DNA was 3,073 shown in SEQ ID NO: 6 in the sequence listing. It was found to contain a base sequence consisting of base pairs. The amino acid sequence deduced from this nucleotide sequence is as described in SEQ ID NO: 6 in the Sequence Listing, and this amino acid sequence was compared with the N-terminal amino acid sequence and the partial amino acid sequence shown in SEQ ID NO: 8, 11, or 12 in the Sequence Listing. However, the N-terminal amino acid sequence shown in SEQ ID NO: 8 is the first to twentieth sequence in SEQ ID NO: 6, and the partial amino acid sequence of SEQ ID NO: 11 or 12 is 606 to 606 in SEQ ID NO: 6 in the sequence listing. The sequence matched the 625th sequence or the 110th to 129th sequences. This is because the enzyme Q36 has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. In Arthrobacter species Q36, the enzyme Q36 is encoded by DNA having the base sequence shown in SEQ ID NO: 4 in the sequence listing. It indicates that
[0079]
【The invention's effect】
As described above, the present invention is based on the discovery of a completely novel enzyme which produces a non-reducing saccharide having a trehalose structure at its terminal from a reducing starch saccharide having a glucose polymerization degree of 3 or more. . The present invention opens the way to produce such enzymes on a large scale and efficiently by recombinant DNA technology. In addition, the enzyme produced by the transformant according to the present invention is an enzyme whose entire amino acid sequence has been clarified, and is a nonreducing saccharide having a trehalose structure at the terminal or trehalose presumed to be used in food or the like. It can be used with confidence in the manufacture of.
[0080]
The present invention is a significant invention having such remarkable functions and effects, and it can be said that it is an invention that greatly contributes to the art.
[0081]
[Sequence list]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[Brief description of the drawings]
FIG. 1 is a diagram showing the optimum temperature of enzyme M-11.
FIG. 2 is a diagram showing an optimum temperature of enzyme Q36.
FIG. 3 is a graph showing the optimum pH of enzyme M-11.
FIG. 4 is a graph showing the optimum pH of enzyme Q36.
FIG. 5 is a diagram showing the thermal stability of enzyme M-11.
FIG. 6 is a graph showing the thermal stability of enzyme Q36.
FIG. 7 is a graph showing the pH stability of enzyme M-11.
FIG. 8 is a graph showing pH stability of enzyme Q36.
FIG. 9 is a restriction map of pBMT7 which is a recombinant DNA according to the present invention. In addition, in the figure, the thick line display part shows the DNA encoding the enzyme M-11.
FIG. 10 is a restriction map of pBQT13 which is a recombinant DNA according to the present invention. In the figure, the bold line indicates the DNA encoding the enzyme Q36.

Claims (11)

The end of glucose polymerization degree of 3 or more reducing starch sugar generates non-reducing saccharides having a trehalose structure, or the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing, or its enzymatic activity in a range that holds DNA encoding an enzyme having an amino acid sequence in which one or more of the amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 in the sequence listing is substituted, deleted, or added to another amino acid. The DNA according to claim 1, wherein the enzyme has the following physicochemical properties.
(1) Molecular weight of about 76,000 to 87,000 daltons (SDS-polyacrylamide gel electrophoresis)
(2) Isoelectric point of about 3.6 to 4.6 (isoelectric focusing) DNA Do nucleotide sequence shown in SEQ ID NO: 3 or 4 in the Sequence Listing, one base in SEQ ID NO: 3 or 4 of the base sequence in the sequence listing the enzymatic activity of an enzyme that encodes the extent that they hold, or two or more substituents The DNA according to claim 1 or 2, which has a base sequence deleted, deleted or added, or a base sequence complementary thereto. Based on the degeneracy of the genetic code, without changing the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing, one or more bases in the base sequence shown in SEQ ID NO: 3 or 4 in the sequence listing are replaced with other bases The DNA according to any one of claims 1 to 3, wherein the DNA is replaced by: The DNA according to any one of claims 1 to 4, which has the nucleotide sequence shown in SEQ ID NO: 5 or 6 in the sequence listing. The DNA according to any one of claims 1 to 5, which is derived from a microorganism belonging to the genus Rhizobium or the genus Arthrobacter. A replicable recombinant DNA comprising the DNA according to any one of claims 1 to 6 and a vector capable of autonomous replication. The replicable recombinant DNA according to claim 7, wherein the autonomously replicable vector is a plasmid vector Bluescript II SK (+). A transformant obtained by appropriately introducing the replicable recombinant DNA according to claim 7 or 8 into a host. The transformant according to claim 9, wherein the host is Escherichia coli. A DNA fragment comprising the DNA according to any one of claims 1 to 6, which encodes an enzyme that generates a non-reducing saccharide having a trehalose structure at a terminal from a reducing starch saccharide having a glucose polymerization degree of 3 or more.

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