JP3557276B2 - DNA encoding an enzyme, recombinant DNA containing the same, and transformant - Google Patents

Description

【００２５】
【表２】

【００２６】
表１及び表２に示すように、酵素Ｍ−１１及び酵素Ｑ３６は、末端にトレハロース構造を有するグルコース重合度３以上の非還元性糖質からトレハロースとグルコース又はマルトオリゴ糖をほぼ定量的に遊離した。一方、グルコース重合度３以上のマルトオリゴ糖を基質にすると、両酵素とも全く作用を示さなかった。これらの事実は、当該酵素が末端にトレハロース構造を有するグルコース重合度３以上の非還元性糖質に選択的に作用し、そのトレハロース残基とグリコシル残基間のグリコシド結合を特異的に加水分解することを示唆している。斯かる酵素作用は未だ報告されておらず、全く新規な作用機序を辿るものと推定される。
【００２７】
【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５７，０００乃至６８，０００ダルトンに相当する位置に単一バンドを示した。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。
【００２８】
【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３．３乃至４．６に等電点を示した。
【００２９】
【実験例２−４ 至適温度】
常法により、５０ｍＭ燐酸緩衝液（ｐＨ７．０）中で３０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４５℃付近に至適温度を示した。
【００３０】
【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３０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、ｐＨ６．０乃至７．５付近に至適ｐＨを示した。
【００３１】
【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、３０乃至４５℃付近まで安定であった。
【００３２】
【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１０．０付近まで安定であった。
【００３３】
【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。
【００３４】
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。
【００３５】
【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、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４３分後又は約５７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」と言う。）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、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。
【００３６】
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４分後又は約２４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」と言う。）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。
【００３７】
以上のような理化学的性質を有する酵素は未だ知られておらず、新規物質であると判断される。なお、リゾビウム・スピーシーズＭ−１１は岡山県岡山市の土壌から分離され、平成４年１２月２４日以降、茨城県つくば市東１丁目１番３号にある通商産業省工業技術院生命工学工業技術研究所、特許微生物寄託センターに寄託番号『ＦＥＲＭ ＢＰ−４１３０』で寄託されている。一方、アルスロバクター・スピーシーズＱ３６は岡山県総社市の土壌から分離されたものであり、平成５年６月３日以降、同センターに寄託番号『ＦＥＲＭ ＢＰ−４３１６』で寄託されている。特開平７−２１３２８３号公報（特願平５−３４０３４３号明細書）には、酵素の性質・性状とともに、両微生物の菌学的性質が詳細に開示されている。
【００３８】
そこで、本発明者が、実験例２−８又は２−９で明らかにした酵素Ｍ−１１の部分アミノ酸配列に基づき化学合成したオリゴヌクレオチドをプローブにし、リゾビウム・スピーシーズＭ−１１の染色体ＤＮＡを鋭意検索したところ、下記の配列表における配列番号３に示す塩基配列を有する１，７６７塩基対からなるＤＮＡ断片が得られた。そして、その塩基配列を解読したところ、酵素Ｍ−１１は５８９個のアミノ酸からなる、配列表における配列番号１に示すアミノ酸配列を有していることが判明した。
【００３９】
一方、酵素Ｑ３６の部分アミノ酸配列に基づき化学合成したオリゴヌクレオチドをプローブにし、アルスロバクター・スピーシーズＱ３６の染色体ＤＮＡを同様に検索したところ、配列表における配列番号４に示す塩基配列を有する１，７９１塩基対からなるＤＮＡ断片が得られた。この塩基配列を解読したところ、酵素Ｑ３６は５９７個のアミノ酸からなり、配列表における配列番号２に示すアミノ酸配列を有していることが判明した。
【００４０】
配列表における配列番号１乃至４に示す塩基配列及びアミノ酸配列を解明するに到った一連の工程を要約すると、次のようになる。
（１） 供与体微生物の培養物から当該酵素を分離し、高度に精製した。精製酵素をプロテアーゼにより部分加水分解後、加水分解物より２種類のペプチド断片を単離し、そのアミノ酸配列を決定した。
（２） 別途、供与体微生物の菌体より染色体ＤＮＡを分離し、精製後、制限酵素により部分的に切断した約２，０００乃至６，０００塩基対からなるＤＮＡ断片を採取した。ＤＮＡリガーゼにより、このＤＮＡ断片を予め制限酵素で切断しておいたプラスミドベクターに連結し、組換えＤＮＡを作製した。
（３） 大腸菌に組換えＤＮＡを導入して形質転換体を作製し、前記部分アミノ酸配列に基づき化学合成したオリゴヌクレオチドをプローブとするコロニーハイブリダイゼーションにより当該酵素をコードするＤＮＡを含む形質転換体を選択した。
（４） 形質転換体から組換えＤＮＡを採取し、プライマーとともにアニーリング後、ＤＮＡポリメラーゼを作用させてプライマーを伸長し、得られた相補鎖ＤＮＡをジデオキシ・チェーン・ターミネータ法により分析して塩基配列を決定した。そして、その塩基配列から推定されるアミノ酸配列と前記部分アミノ酸配列とを比較し、その塩基配列が当該酵素をコードしていることを確認した。
【００４１】
両供与体微生物の遺伝子がコードするアミノ酸配列は配列表における配列番号１又は２に示すとおりであるが、この発明のＤＮＡは、配列表における配列番号１又は２に示すとおりのアミノ酸配列をコードするものは無論のこと、これらと相同的なアミノ酸配列を有するものをも包含するものとする。すなわち、組換えＤＮＡ技術の進歩により、斯界においては、酵素の作用を実質的に変えることなく、比較的容易にその構成アミノ酸の１個又は２個以上を他のアミノ酸で置換できるようになった。また、同じＤＮＡであっても、それを導入する宿主や、そのＤＮＡを含む形質転換体の培養に使用する栄養培地の成分・組成、培養温度・ｐＨなどに依っては、宿主内酵素によるＤＮＡ発現後の修飾などにより、所期の酵素作用は保持しているものの、配列表における配列番号１又は２のアミノ酸配列におけるＮ末端付近のアミノ酸の１個又２個以上が欠失したり、Ｎ末端に１個又は２個以上のアミノ酸が新たに付加した変異体の産生することがある。斯かる技術水準に鑑み、この発明でいう酵素とは、配列表における配列番号１又は２に示すアミノ酸配列をそのまま具備するものは言うに及ばず、そのアミノ酸配列におけるアミノ酸の１個又は２個以上が他のアミノ酸に置き換わるか、欠失若しくは付加した変異体であっても、それが末端にトレハロース構造を有するグルコース重合度３以上の非還元性糖質からトレハロースを遊離するかぎり包含するものとする。
【００４２】
さらに、斯界においては、遺伝子コードの縮重により、コードするアミノ酸配列を変えることなく、ＤＮＡにおける塩基の１個又は２個以上を他の塩基で置換することができる。これにより、この発明のＤＮＡは、配列表における配列番号３又は４に示す塩基配列をそのまま有するもののみならず、それが配列表における配列番号１又は２に示すアミノ酸配列を有する酵素若しくはその相同変異体をコードするものであるかぎり、遺伝子コードの縮重に基づき、塩基の１個又は２個以上が他の塩基に置き換わったものをも包含するものとする。
【００４３】
また、現在の組換えＤＮＡ技術に依るときには、一般に５′末端からの塩基配列が決まれば、これに相補的な塩基配列は一義的に定まる。したがって、この発明のＤＮＡは、上記いずれかの塩基配列に相補的な塩基配列を有するものも包含するものとする。なお、この発明のＤＮＡが宿主中で実際に当該酵素の産生を発現するために、当該酵素又はその相同変異体をコードする塩基配列における塩基の１個又は２個以上を他の塩基で適宜置換し得ることは言うまでもない。
【００４４】
この発明のＤＮＡは上記のごときものであるが、この発明のＤＮＡは、それが前記のごとき配列を有するかぎり、それが天然に由来するものか人為的に合成されたものであるかは問わない。天然の給源としては、例えば、リゾビウム・スピーシーズＭ−１１（ＦＥＲＭ ＢＰ−４１３０）、アルスロバクター・スピーシーズＱ３６（ＦＥＲＭ ＢＰ−４３１６）、ブレビバクテリウム・ヘロボルム（ＡＴＣＣ１１８２２）及びミクロコッカス・ロゼウス（ＡＴＣＣ１８６）を含むリゾビウム属、アルスロバクター属、ブレビバクテリウム属、ミクロコッカス属の微生物が挙げられ、これら微生物の菌体からはこの発明のＤＮＡを含む遺伝子が得られる。すなわち、斯かる微生物を栄養培地に植菌し、好気的条件下で約１乃至３日間培養後、培養物から菌体を採取し、リゾチームやβ−グルカナーゼなどの細胞壁溶解酵素や超音波で処理することにより当該ＤＮＡを含む遺伝子を菌体外に溶出させる。このとき、細胞壁溶解酵素にプロテアーゼなどの蛋白質加水分解酵素を併用したり、菌体を超音波処理する際、ＳＤＳなどの界面活性剤を共存させたり凍結融解してもよい。斯くして得られる処理物に、例えば、フェノール抽出、アルコール沈澱、遠心分離、プロテアーゼ処理、リボヌクレアーゼ処理などの斯界における通常一般の方法を適用すれば目的のＤＮＡが得られる。
【００４５】
一方、この発明のＤＮＡを人為的に合成するには、例えば、配列表における配列番号３又は４に示す塩基配列に基づいて化学合成するか、配列表における配列番号１又は２に示すアミノ酸配列をコードするＤＮＡを自律複製可能な適宜ベクターに挿入して組換えＤＮＡとし、これを適宜宿主に導入して得られる形質転換体を培養し、培養物から菌体を採取し、その菌体から当該ＤＮＡを含むプラスミドを採取すればよい。
【００４６】
さて、この発明は、本来、当該酵素を産生しないけれども、比較的容易に増殖させることのできる微生物や動植物に導入すると当該酵素の産生を発現する複製可能な組換えＤＮＡに係わるものでもある。斯かる組換えＤＮＡは、通常、前述のごときＤＮＡと自律複製可能なベクターを含んでなり、ＤＮＡが入手できれば、通常一般の方法により比較的容易に調製することができる。斯かるベクターの例としては、ｐＢＲ３２２、ｐＵＣ１８、Ｂｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）、ｐＵＢ１１０、ｐＴＺ４、ｐＣ１９４、ｐＨＶ１４、ＴＲｐ７、ＹＥｐ７、ｐＢＳ７などのプラスミドベクターやλｇｔ・λＣ、λｇｔ・λＢ、ρ１１、φ１、φ１０５などのファージベクターが挙げられ、このうち、この発明のＤＮＡを大腸菌で発現させるにはｐＢＲ３２２、ｐＵＣ１８、ＢｌｕｅｓｃｒｉｐｔＩＩ ＳＫ（＋）、λｇｔ・λＣ及びλｇｔ・λＢが好適であり、一方、枯草菌で発現させるにはｐＵＢ１１０、ｐＴＺ４、ｐＣ１９４、ρ１１、φ１及びφ１０５が好適である。ｐＨＶ１４、ＴＲｐ７、ＴＥｐ７及びｐＢＳ７は、組換えＤＮＡを２種以上の宿主内で増殖させる場合に有用である。
【００４７】
斯かるベクターにこの発明のＤＮＡを挿入するには、斯界において通常一般の方法が採用される。具体的には、先ず、この発明のＤＮＡを含む遺伝子と自律複製可能なベクターとを制限酵素及び／又は超音波により切断し、次に、生成したＤＮＡ断片とベクター断片とを連結する。遺伝子及びベクターの切断にヌクレオチドに特異的に作用する制限酵素、とりわけ、ＩＩ型の制限酵素、詳細には、Ｓａｕ ３ＡＩ、Ｅｃｏ ＲＩ、Ｈｉｎｄ ＩＩＩ、Ｂａｍ ＨＩ、Ｓａｌ Ｉ、Ｘｂａ Ｉ、Ｓａｃ Ｉ、Ｐｓｔ Ｉなどを使用すれば、ＤＮＡ断片とベクター断片を連結するのが容易となる。ＤＮＡ断片とベクター断片を連結するには、必要に応じて、両者をアニーリングした後、生体内又は生体外でＤＮＡリガーゼを作用させればよい。斯くして得られる組換えＤＮＡは、適宜宿主に導入して形質転換体とし、これを培養することにより無限に複製可能である。
【００４８】
この発明による組換えＤＮＡは、大腸菌、枯草菌、放線菌、酵母を始めとする適宜の宿主微生物に導入することができる。宿主が大腸菌の場合には、宿主を組換えＤＮＡとカルシウムイオンの存在下で培養すればよく、一方、宿主が枯草菌の場合には、コンピテントセル法やプロトプラスト法を適用すればよい。形質転換体をクローニングするには、コロニーハイブリダイゼーション法を適用するか、末端にトレハロース構造を有するグルコース重合度３以上の非還元性糖質を含む栄養培地で培養し、該非還元性糖質よりトレハロースを遊離するものを選択すればよい。
【００４９】
斯くして得られる形質転換体は、栄養培地で培養すると、菌体内外に当該酵素を産生する。栄養培地には、通常、炭素源、窒素源、ミネラル、さらには、必要に応じて、アミノ酸やビタミンなどの微量栄養素を補足した通常一般の液体培地が使用され、個々の炭素源としては、例えば、澱粉、澱粉加水分解物、グルコース、果糖、蔗糖などの糖質が、また、窒素源としては、例えば、アンモニア若しくはアンモニウム塩、尿素、硝酸塩、ペプトン、酵母エキス、脱脂大豆、コーンスティープリカー、肉エキスなどの含窒素無機乃至有機物が挙げられる。形質転換体を斯かる栄養培地に植菌し、栄養培地を温度２５乃至６５℃、ｐＨ２乃至８に保ちつつ、通気撹拌などによる好気的条件下で約１乃至６日間培養すれば、当該酵素を含む培養物が得られる。この培養物は酵素剤としてそのまま使用可能ではあるが、通常は使用に先立ち、必要に応じて、超音波や細胞壁溶解酵素により菌体を破砕した後、濾過、遠心分離などにより酵素を菌体又は菌体破砕物から分離し、精製する。精製には酵素を精製するための通常一般の方法が採用でき、例えば、菌体又は菌体破砕物を除去した培養物に濃縮、塩析、透析、分別沈澱、ゲル濾過クロマトグラフィー、イオン交換クロマトグラフィー、疎水クロマトグラフィー、アフィニティークロマトグラフィー、ゲル電気泳動、等電点電気泳動などの１種若しくは２種以上を適宜組合せて適用すればよい。
【００５０】
前述のとおり、当該酵素は、末端にトレハロース構造を有するグルコース重合度３以上の非還元性糖質からトレハロースを遊離するという、従来の酵素には見られない顕著な作用を有する。当該酵素のこの作用を利用することにより、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、α−マルトペンタオシルトレハロースなどの一連の非還元性糖質からトレハロースを収量良く、効率的に得ることができる。これら非還元性糖質は、特開平７−１４３８７６号公報（特願平５−３４９２１６号明細書）に開示された非還元性糖質生成酵素を使用することにより、澱粉又はアミロース、アミロペクチンなどの澱粉質に酸及び／又はアミラーゼを作用して得られる澱粉加水分解物から好収量で得ることができる。したがって、当該酵素と特開平７−１４３８７６号公報（特願平５−３４９２１６号明細書）に開示された非還元性糖質生成酵素を組合せて使用することにより、澱粉又は澱粉質を原料に、従来大量入手の難しかったトレハロースが比較的容易に所望量得られることとなる。
【００５１】
以下、２〜３の実施例に基づき、この発明を具体的に説明するが、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。
【００５２】
【実施例１ リゾビウム・スピーシーズＭ−１１由来のＤＮＡを含む組換えＤＮＡと形質転換体の調製】
【００５３】
【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。
【００５４】
【実施例１−２ 組換えＤＮＡ ｐＢＭＵ２７と形質転換体ＢＭＵ２７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２，０００乃至６，０００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。
【００５５】
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６，０００個のコロニーをナイロン膜上に固定した。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８乃至１３番目のＰｈｅ−Ａｓｐ−Ｉｌｅ−Ｔｒｐ−Ａｌａ−Ｐｒｏで表される配列に基づき５′−ＴＴＹＧＡＹＡＴＨＴＧＧＧＣＮＣＣ−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１４種類の形質転換体を選択した。
【００５６】
その後、常法により、これら１４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２乃至６番目のＡｓｐ−Ｔｒｐ−Ａｌａ−Ｇｌｕ−Ａｌａで表される配列に基づき化学合成した５′−ＧＡＹＴＧＧＧＣＮＧＡＲＧＣ−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、『ｐＢＭＵ２７』又は『ＢＭＵ２７』と命名した。
【００５７】
上記で得た形質転換体ＢＭＵ２７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 ｐＢＭＵ２７は約５，７００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１，７６７塩基対からなるＤＮＡは、制限酵素Ｅｃｏ ＲＶ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。
【００５８】
【実施例１−３ 形質転換体ＢＭＵ２７による酵素の産生】
松谷化学工業製澱粉加水分解物『パインデックス＃４』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ＢＭＵ２７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４，０００単位の酵素が産生していた。
【００５９】
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組成の液体培地に植菌し、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２，０００単位と、形質転換体ＢＭＵ２７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。
【００６０】
その後、形質転換体ＢＭＵ２７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５７，０００乃至６８，０００ダルトンを、また、等電点電気泳動で約３．３乃至４．６に等電点を示すなど、酵素Ｍ−１１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。
【００６１】
【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 ｐＢＭＵ２７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２０ｍＭ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。
【００６２】
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、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９８％（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。反応物を沸騰水溶中で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。
【００６３】
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２，１６１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８乃至２７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１０乃至３０番目又は第４９３乃至５０９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の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。
【００６４】
【実施例３ アルスロバクター・スピーシーズＱ３６由来のＤＮＡを含む組換えＤＮＡと形質転換体の調製】
【００６５】
【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。
【００６６】
【実施例３−２ 組換えＤＮＡ ｐＢＲＴ３２と形質転換体ＢＲＴ３２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２，０００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５，０００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１１に示すアミノ酸配列における第５乃至１０番目のＭｅｔ−Ｇｌｙ−Ｔｒｐ−Ａｓｐ−Ｐｒｏ−Ａｌａで表される配列に基づき５′−ＡＴＧＧＧＮＴＧＧＧＡＹＣＣＮＧＣ−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１０種類の形質転換体を選択した。
【００６７】
実施例１−２と同様にして、これら１０種類の形質転換体から組換えＤＮＡを採取し、これに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８乃至１２番目のＴｙｒ−Ａｓｐ−Ｖａｌ−Ｔｒｐ−Ａｌａで表される配列に基づき化学合成した５′−ＴＡＹＧＡＹＧＴＮＴＧＧＧＣ−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、『ｐＢＲＴ３２』又は『ＢＲＴ３２』と命名した。
【００６８】
その後、この形質転換体ＢＲＴ３２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 ｐＢＲＴ３２は約６，２００塩基対からなり、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１，７９１塩基対からなるＤＮＡは、制限酵素Ｋｐｎ 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。
【００６９】
【実施例３−３ 形質転換体ＢＲＴ３２による酵素の産生】
松谷化学工業製澱粉加水分解物『パインデックス＃４』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ＢＲＴ３２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３，９００単位の酵素が産生していた。
【００７０】
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１，８００単位と、形質転換体ＢＲＴ３２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。
【００７１】
その後、形質転換体ＢＲＴ３２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５７，０００乃至６８，０００ダルトンを、また、等電点電気泳動で約３．３乃至４．６に等電点を示すなど、酵素Ｑ３６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。
【００７２】
【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 ｐＢＲＴ３２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２，０５６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の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２乃至２１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４７０乃至４８９番目又は第１１乃至３０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の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。
【００７３】
【発明の効果】
以上説明したように、この発明は、末端にトレハロース構造を有するグルコース重合度３以上の非還元性糖質からトレハロースを遊離する、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の製造に安心して使用し得るものである。
【００７４】
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。
【００７５】
【配列表】

【００７６】

【００７７】

【００７８】

【００７９】

【００８０】

【００８１】

【００８２】

【００８３】

【００８４】

【００８５】

【００８６】

【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 ｐＢＭＵ２７の制限酵素地図を示す図である。図中、太線で表示した部分は酵素Ｍ−１１をコードするＤＮＡである。
【図１０】この発明による組換えＤＮＡ ｐＢＲＴ３２の制限酵素地図を示す図である。図中、太線で表示した部分は酵素Ｑ３６をコードするＤＮＡである。[0001]
[Industrial applications]
The present invention relates to a novel DNA encoding an enzyme that releases trehalose from a non-reducing saccharide having a trehalose structure at the terminal and having a degree of polymerization of glucose of 3 or more, a recombinant DNA containing the DNA, and a transformant. .
[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 has hardly been actually 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 conducted an extensive 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 carbohydrate having a trehalose structure at the terminal from three or more reducing starch sugars, is produced. 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. It was found to be hydrolyzed to sugar. 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 these enzymes, and when large-scale production of trehalose or a non-reducing saccharide having a trehalose structure at a terminal is attempted, 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 releases trehalose from a non-reducing saccharide having a trehalose structure at a terminal.
[0007]
Another object of the present invention is to provide a replicable recombinant DNA containing the DNA.
[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 releases trehalose from a non-reducing saccharide having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more.
[0010]
The present invention solves the second problem by providing a DNA encoding an enzyme that releases trehalose from a non-reducing saccharide having a trehalose structure at the terminal and having a degree of polymerization of glucose of 3 or more, and a replicable vector comprising an autonomously replicable vector. The problem is solved by a simple recombinant DNA.
[0011]
The present invention provides a third object of the present invention to provide a recombinant comprising a DNA encoding an enzyme that releases trehalose from a non-reducing carbohydrate having a terminal trehalose structure and having a degree of polymerization of glucose of 3 or more, and a vector capable of autonomous replication. The problem is solved by a transformant obtained by appropriately introducing 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. The recombinant DNA is used in a host which 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]
The present invention is based on the discovery of a completely novel enzyme which releases trehalose from a non-reducing saccharide having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more. Such enzymes can be obtained from cultures of Rhizobium species M-11 or Arthrobacter species Q36, and the present inventors have isolated the enzyme by combining various purification methods, mainly column chromatography, Examination of its properties and properties revealed that the essence was a polypeptide and had the following physicochemical properties.
(1) Action
Trehalose is released from non-reducing saccharides having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more.
(2) Molecular weight
Approximately 57,000 to 68,000 daltons (SDS-polyacrylamide gel electrophoresis)
(3) Isoelectric point
Approx. 3.3 to 4.6 (isoelectric focusing)
(4) Optimal temperature
Incubation at pH 7.0 for 30 minutes shows an optimum temperature around 35 to 45 ° C.
(5) Optimum pH
When incubated at 40 ° C. for 30 minutes, it shows an optimum pH around pH 6.0 to 7.5.
(6) Thermal stability
When incubated at pH 7.0 for 60 minutes, it is stable up to around 30 to 45 ° C.
(7) pH stability
When incubated at 25 ° C. for 16 hours, the pH is stable up to around 5.5 to 10.0.
[0016]
Next, an experiment was conducted to elucidate the physicochemical properties of the enzymes produced by Rhizobium species M-11 or Arthrobacter species Q36 (hereinafter referred to as "enzyme M-11" or "enzyme Q36", respectively). Will be described.
[0017]
[Experimental example 1 Purification of enzyme]
[0018]
Experimental Example 1-1 Purification of Enzyme M-11
In a 500 ml Erlenmeyer flask, 2.0% (w / v) of a starch hydrolyzate “Paindex # 4” manufactured by Matsutani Chemical Industry, 0.5% (w / v) of peptone, and 0.1% (w / v) of yeast extract ), 100 ml of a liquid medium (pH 7.0) containing 0.1% (w / v) of disodium hydrogen phosphate and 0.1% (w / v) of potassium dihydrogen phosphate, and autoclaved at 120 ° C for 20 minutes. And sterilized. 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.
[0019]
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 enzyme M-11 thus purified was about 240 units / mg protein, and the yield was about 650 units per liter of culture.
[0020]
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) of α-maltotriosyltrehalose is added, 1 ml of the enzyme solution is added thereto, and the mixture is incubated at 40 ° C. for 30 minutes to react. . Then, 1 ml of the reaction solution is taken and added to 2 ml of a Somogi copper solution to stop the reaction, and then the reducing power is measured by the Somogi-Nelson method. As a control, an enzyme previously inactivated by heating at 100 ° C. for 10 minutes is treated in the same manner as described above. One unit of the enzyme is defined as the amount of the enzyme that increases the reducing power corresponding to 1 μmol of glucose per minute under the above conditions.
[0021]
[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 450 units / mg protein was obtained at a yield of about 650 units per liter of culture. Obtained.
[0022]
[Experimental example 2 Physicochemical properties of enzyme]
[0023]
[Experimental example 2-1 Action]
JP-A-7-143876 ( Japanese Patent Application No. 5-349216 ) Non-reducing compositions containing at least 98% of solids per unit of α-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose, α-maltotetraosyltrehalose or α-maltopentaosyltrehalose by the method disclosed in Sex sugars were prepared. Then, using either of them as a substrate, it is dissolved in 50 mM phosphate buffer (pH 7.0) to a concentration of 20% (w / v), and the purified enzyme M-11 or Q36 obtained in Experimental Example 1 is added to the solution. Two units were added 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. Separately, an aqueous solution containing any of high-purity maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose in place of the non-reducing carbohydrate is treated in the same manner as described above, and the treated product is analyzed. As a control. Tables 1 and 2 show the sugar composition of the substrate and the reactant when M-11 and Q36 were added, respectively.
[0024]
[Table 1]

[0025]
[Table 2]

[0026]
As shown in Tables 1 and 2, enzyme M-11 and enzyme Q36 almost quantitatively released trehalose and glucose or maltooligosaccharide from non-reducing saccharides having a trehalose structure at the terminal and having a degree of polymerization of glucose of 3 or more. . On the other hand, when a maltooligosaccharide having a glucose polymerization degree of 3 or more was used as a substrate, neither enzyme showed any action. These facts indicate that the enzyme selectively acts on non-reducing saccharides having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more, and specifically hydrolyzes glycosidic bonds between trehalose residues and glycosyl residues. Suggests that you Such an enzymatic action has not been reported yet, and is presumed to follow a completely novel mechanism of action.
[0027]
[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). The enzyme Q36 also showed a single band at a position corresponding to a molecular weight of about 57,000 to 68,000 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).
[0028]
[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 3.3 to 4.6.
[0029]
[Experimental example 2-4 Optimum temperature]
According to a conventional method, the test was performed under the condition of incubating for 30 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 45 ° C. The optimum temperature was indicated.
[0030]
[Experimental example 2-5 Optimum pH]
According to a conventional method, the test was performed under the conditions of incubation at 40 ° C. for 30 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 Enzyme M-11 and Enzyme Q36 showed optimum pH around pH 6.0 to 7.5.
[0031]
[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 30 to 45 ° C. It was stable.
[0032]
[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 10.0.
[0033]
[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.
[0034]
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.
[0035]
[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 / min. The solution was passed at a flow rate. Then, a fraction containing the peptide fragment (hereinafter, referred to as “peptide fragment A” or “peptide fragment B,” respectively) eluted about 43 minutes or about 57 minutes after the start of the passage of the solution, was collected, dried in vacuo, 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.
[0036]
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 a peptide fragment (hereinafter, referred to as “peptide fragment C” or “peptide fragment D”) eluted about 4 minutes or about 24 minutes after the start of the flow 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.
[0037]
Enzymes having the above 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 It has been deposited at the research institute and 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. JP-A-7-213283 ( Japanese Patent Application No. 5-340343 ) Discloses in detail the mycological properties of both microorganisms as well as the properties and properties of the enzymes.
[0038]
Thus, the present inventors used the oligonucleotide chemically synthesized based on the partial amino acid sequence of the enzyme M-11 clarified in Experimental Example 2-8 or 2-9 as a probe and eagerly studied the chromosomal DNA of Rhizobium species M-11. As a result of the search, a DNA fragment consisting of 1,767 base pairs having the base sequence shown in SEQ ID NO: 3 in the following sequence listing was obtained. Then, when the base sequence was decoded, it was found that the enzyme M-11 had an amino acid sequence consisting of 589 amino acids and represented by SEQ ID NO: 1 in the sequence listing.
[0039]
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 species Q36 was similarly searched, and it was found that 1,791 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 enzyme Q36 was composed of 597 amino acids and had the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
[0040]
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 partially hydrolyzing 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 then a DNA fragment consisting of about 2,000 to 6,000 base pairs, which was partially digested with restriction enzymes, was collected. 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.
[0041]
The amino acid sequences encoded by the genes of both donor microorganisms are as shown in SEQ ID NO: 1 or 2 in the Sequence Listing, and the DNA of the present invention encodes the amino acid sequence as shown in SEQ ID NO: 1 or 2 in the Sequence Listing. Of course, 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. Although the desired enzymatic action is retained by modification after expression, 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 to the terminal. 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 replaced by another amino acid, or is a deletion or addition mutant, as long as it releases trehalose from a non-reducing saccharide having a terminal trehalose structure and a glucose polymerization degree of 3 or more. .
[0042]
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.
[0043]
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. Needless to say, it can be done.
[0044]
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. . Natural sources include, for example, Rhizobium species M-11 (FERM BP-4130), Arthrobacter species Q36 (FERM BP-4316), Brevibacterium herovolum (ATCC 11822) and Micrococcus roseus (ATCC 186). And microorganisms belonging to the genera Rhizobium, Arthrobacter, Brevibacterium, and Micrococcus, and a gene containing the DNA of the present invention can be obtained from the cells of these microorganisms. 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 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.
[0045]
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 the resulting transformant is appropriately introduced into a host, followed by culturing a transformant, collecting cells from the culture, and culturing the cells from the cells. A plasmid containing DNA may be collected.
[0046]
The present invention also relates to a replicable recombinant DNA which expresses the production of the enzyme when introduced into a microorganism or animal or plant which does not naturally 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 autonomously replicating, and if the DNA is available, it can be prepared relatively easily by ordinary methods. 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 suitable for expressing the DNA of the present invention in Escherichia coli, while expression in Bacillus subtilis is preferred. For this purpose, pUB110, pTZ4, pC194, ρ11, φ1, and φ105 are preferable. pHV14, TRp7, TEp7, and pBS7 are useful when growing recombinant DNA in more than one host.
[0047]
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.
[0048]
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 the transformant, a colony hybridization method is applied, or the transformant is cultured in a nutrient medium containing a non-reducing saccharide having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more, and trehalose is obtained from the non-reducing saccharide. May be selected as those that release
[0049]
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.
[0050]
As described above, the enzyme has a remarkable action, which is not found in conventional enzymes, that releases trehalose from a non-reducing saccharide having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more. By utilizing this action of the enzyme, a series of non-reducing processes such as α-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose, α-maltotetraosyltrehalose, α-maltopentaosyltrehalose, etc. Trehalose can be obtained efficiently and efficiently from saccharides. These non-reducing sugars are JP-A-7-143876 ( Japanese Patent Application No. 5-349216 ) By using the non-reducing carbohydrate-forming enzyme disclosed in the above, a starch or a starch such as amylose or amylopectin can be obtained in good yield from a starch hydrolyzate obtained by the action of an acid and / or an amylase. . Therefore, the enzyme and JP-A-7-143876 ( Japanese Patent Application No. 5-349216 ) By using the non-reducing carbohydrate-forming enzyme disclosed in the above in combination, a desired amount of trehalose, which has conventionally been difficult to obtain in large quantities, can be obtained relatively easily from starch or starchy materials.
[0051]
Hereinafter, the present invention will be specifically described based on a few examples. The technique itself used in these examples is well known in the art, and is described in, for example, J. Sambrook et al. Laboratory Manual, 2nd edition, 1989, published by Cold Spring Harbor Laboratory Press.
[0052]
Example 1 Preparation of Recombinant DNA Containing DNA from Rhizobium sp. M-11 and Transformant
[0053]
[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.
[0054]
Example 1-2 Preparation of Recombinant DNA pBMU27 and Transformant BMU27
About 1 ml of the purified chromosomal DNA solution obtained in Example 1-1 was taken, about 35 units of the restriction enzyme Sau3AI was added thereto, and the mixture was allowed to react at 37 ° C. for about 20 minutes to partially cut the chromosomal DNA. A DNA fragment consisting of about 2,000 to 6,000 base pairs was collected by centrifugation. 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.
[0055]
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. A nylon membrane was placed thereon, and about 6,000 colonies formed on the medium were fixed on the nylon membrane. Separately, based on the sequence represented by the 8th to 13th Phe-Asp-Ile-Trp-Ala-Pro in the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, 5'-TTYGAYAATHTGGGCNCC-3 ' Probe 1 of the base sequence to be ³² After labeling with P, hybridization was carried out to colonies of the transformants fixed on the nylon membrane, and 14 types of transformants in which remarkable association was recognized were selected.
[0056]
Thereafter, recombinant DNA was collected from these 14 types of transformants by a conventional method and represented by the second to sixth Asp-Trp-Ala-Glu-Ala in the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing. Probe 2 of the base sequence represented by 5'-GAYTGGCNGARGC-3 ', which was chemically synthesized based on the sequence shown in FIG. 1, was prepared by EM Southern, "Journal of Molecular Biology", Vol. 98, pp. 503-517 ( 1975), and a recombinant DNA exhibiting a remarkable association with probe 2 was selected. The recombinant DNA and transformant selected as described above were named "pBMU27" or "BMU27", respectively.
[0057]
The transformant BMU27 obtained above was inoculated into an L-broth medium (pH 7.0) containing 100 μg / ml of ampicillin, and cultured with rotation and shaking at 37 ° C. for 24 hours. 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 purified product was purified by a conventional method and analyzed. As a result, the recombinant DNA pBMU27 was composed of about 5,700 base pairs and had a structure represented by the restriction map shown in FIG. As shown in FIG. 9, it was found that the DNA consisting of 1,767 base pairs encoding the enzyme M-11 was located downstream near the site of cleavage by the restriction enzyme Eco RV.
[0058]
Example 1-3 Production of Enzyme by Transformant BMU27
Starch hydrolyzate “Paindex # 4” manufactured by Matsutani Chemical Industry 2.0% (w / v), peptone 0.5% (w / v), yeast extract 0.1% (w / v), hydrogen phosphate A liquid medium containing 0.1% (w / v) of sodium and 0.1% (w / v) of potassium dihydrogen phosphate was adjusted to pH 7.0, and 50 μg / ml of ampicillin was added thereto, followed by heat sterilization at 120 ° C. for 20 minutes. Then, after cooling, the transformant BMU27 obtained in Example 1-2 was inoculated, and cultured at 37 ° C. for 24 hours with rotary shaking. The culture was sonicated to disrupt the cells, and the insolubles were removed by centrifugation. After measuring the enzyme activity in the supernatant, the enzyme activity of about 4,000 units per liter of culture was calculated. Was produced.
[0059]
Separately, as a control, Escherichia coli XLI-Blue strain and Rhizobium sp. M-11 were inoculated into a liquid medium having the same composition without ampicillin, and in the case of Rhizobium sp. M-11, 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 Rhizobium species M-11 was about 2,000 units per liter of the culture, which was significantly lower than that of the transformant BMU27. The E. coli XLI-Blue strain used as the host did not produce the enzyme at all.
[0060]
Thereafter, the enzyme produced by the transformant BMU27 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 57,000 to 68,000 daltons by SDS-polyacrylamide gel electrophoresis. Has an isoelectric point of about 3.3 to 4.6 in isoelectric focusing, and has the same physicochemical properties as enzyme M-11. This suggests that the enzyme can be produced by recombinant DNA technology, and that the productivity of the enzyme is also significantly improved.
[0061]
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 pBMU27 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. A primer 1 having a base sequence represented by 5′-GTAAAACGACGGCCAGT-3 ′ chemically synthesized with the template DNA was mixed with 50 pmol / ml and a 40 mM Tris-HCl buffer (pH 7.5) containing 20 mM magnesium chloride and 20 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.
[0062]
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 aqueous sodium chloride solution containing 8 μM of any of ddATP, ddCTP, ddGTP and ddTTP and 80 μM of dNTP was added thereto, and 37 Incubate at 5 ° C. for 5 minutes to react, and a 98% (v / v) aqueous formamide solution containing 20 mM EDTA, 0.05% (w / v) bromophenol blue and 0.05% (w / v) xylene cyanol Was added to stop the reaction. The reaction product was heated in a boiling aqueous solution for 3 minutes, 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.
[0063]
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 2,161 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 8th to 27th sequence in SEQ ID NO: 5, and the partial amino acid sequence of SEQ ID NO: 9 or 10 was SEQ ID NO: 5 in the sequence listing. In the 10th to 30th sequences or the 493th to 509th sequences. This means that the enzyme M-11 has the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and in Rhizobium species M-11, the enzyme M-11 is derived from the DNA having the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing. Indicates that it is coded.
[0064]
Example 3 Preparation of Recombinant DNA Containing DNA from Arthrobacter sp. Q36 and Transformant
[0065]
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.
[0066]
Example 3-2 Preparation of Recombinant DNA pBRT32 and Transformant BRT32
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 2,000 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′-ATGGGNTGGGAYCCNGC-3 ′ based on the sequence represented by the 5th to 10th Met-Gly-Trp-Asp-Pro-Ala in the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing. Probe 3 having the nucleotide sequence represented was chemically synthesized and ³² After labeling with P, the cells were hybridized to colonies of the transformant immobilized on the nylon membrane, and 10 types of transformants in which remarkable association was recognized were selected.
[0067]
In the same manner as in Example 1-2, recombinant DNAs were collected from these 10 kinds of transformants, and the 8th to 12th Tyr-Asp-Val- in the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing were collected therefrom. Probe 4 having a base sequence represented by 5'-TAYGAYGTNTGGGC-3 ', which was chemically synthesized based on the sequence represented by Trp-Ala, was hybridized, and a recombinant DNA showing a remarkable association was selected. The recombinant DNA and transformant selected as described above were named "pBRT32" or "BRT32", respectively.
[0068]
Thereafter, the transformant BRT32 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 pBRT32 was composed of about 6,200 base 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 1,791 base pairs encoding the enzyme Q36 was located downstream near the site of cleavage by the restriction enzyme KpnI.
[0069]
Example 3-3 Enzyme Production by Transformant BRT32
Starch hydrolyzate “Paindex # 4” manufactured by Matsutani Chemical Industry 2.0% (w / v), peptone 0.5% (w / v), yeast extract 0.1% (w / v), hydrogen phosphate A liquid medium containing 0.1% (w / v) of sodium and 0.1% (w / v) of potassium dihydrogen phosphate was adjusted to pH 7.0, and 50 μg / ml of ampicillin was added thereto, followed by heat sterilization at 120 ° C. for 20 minutes. After cooling, the transformant BRT32 obtained in Example 3-2 was inoculated, and cultured at 37 ° C. for 24 hours with rotary 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 was converted to about 3,900 units of enzyme per liter of culture. Was produced.
[0070]
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 1,800 units per liter of the culture, which was significantly lower than that of the transformant BRT32. The E. coli XLI-Blue strain used as the host did not produce the enzyme at all.
[0071]
Thereafter, the enzyme produced by the transformant BRT32 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 determined to be about 57,000 to 68,000 daltons by SDS-polyacrylamide gel electrophoresis. Has an isoelectric point of about 3.3 to 4.6 by isoelectric focusing, and has the same physicochemical properties as enzyme Q36. This suggests that the enzyme can be produced by recombinant DNA technology, and that the productivity of the enzyme is also significantly improved.
[0072]
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 pBRT32 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 Primer 1 and reacted with T7 DNA polymerase to place Primer 1 from the 5 'end to 3'. Extension toward the ends produced complementary DNA strands. As in Example 2, the dideoxy chain terminator method was applied to the complementary strand DNA, and the DNA fragments separated on the radiogram were analyzed. As a result, the complementary strand DNA was 2,056 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 of SEQ ID NO: 8 corresponds to the 2nd to 21st sequence in SEQ ID NO: 6, and the partial amino acid sequence of SEQ ID NO: 11 or 12 corresponds to 470 to 489 in SEQ ID NO: 6. Or the 11th to 30th sequences. This means that the enzyme Q36 has the amino acid sequence of SEQ ID NO: 2 in the sequence listing. In Arthrobacter species Q36, the enzyme Q36 is encoded by the DNA having the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing. It is shown that.
[0073]
【The invention's effect】
As described above, the present invention is based on the discovery of a completely novel enzyme that releases trehalose from a non-reducing saccharide having a terminal trehalose structure and 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 can be used with confidence in the production of trehalose on the premise of its use in foods and the like.
[0074]
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.
[0075]
[Sequence list]

[0076]

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[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[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 shows a restriction map of the recombinant DNA pBMU27 according to the present invention. In the figure, the portion indicated by the thick line is DNA encoding the enzyme M-11.
FIG. 10 shows a restriction map of recombinant DNA pBRT32 according to the present invention. In the figure, the portion indicated by a thick line is DNA encoding the enzyme Q36.

Claims (13)

Terminus liberated trehalose from glucose polymerization degree of 3 or more non-reducing saccharides having a trehalose structure, or the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence table, or in the sequence listing within a range of its enzymatic activity retained 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 is substituted, deleted, or added to another amino acid. The DNA according to claim 1, wherein the enzyme has a molecular weight of about 57,000 to 68,000 daltons (SDS-polyacrylamide gel electrophoresis). The DNA according to claim 1 or 2, wherein the enzyme has an isoelectric point of about 3.3 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 any one of claims 1 to 3, 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 4, wherein the DNA is substituted with: The DNA according to any one of claims 1 to 5, 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 6, 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 7 and an autonomously replicable vector. The replicable recombinant DNA according to claim 8, 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 8 or 9 into a host. The transformant according to claim 10, wherein the host is Escherichia coli. A DNA fragment comprising the DNA according to any one of claims 1 to 7, which encodes an enzyme that releases trehalose from a non-reducing saccharide having a trehalose structure at a terminal and having a degree of polymerization of glucose of 3 or more. A recombinant DNA comprising the DNA fragment of claim 12.

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