JP4021123B2 - New production method of raffinose - Google Patents

Description

【００２０】
（３）至適ｐＨ及び安定ｐＨ範囲
図３に、本酵素のｐＨに対する影響を示した。安定性の検討は、酵素溶液を各ｐＨの緩衝液中で２４時間、４℃で保持した後、溶液をｐＨ５．５の緩衝液で置換し、基質をＯＮＰα−ガラクトシドを用いて、正反応の酵素活性を定法に従い測定した。その結果、本酵素の至適ｐＨは５．５であり、ｐＨ５．０から１１．０で安定であった。
【００２１】
（４）力価の測定法
本酵素活性は、ＯＮＰＧ（ｏ−ニトロフェニルα−Ｄ−ガラクトピラノシド）法に従って測定した。酵素反応は、０．１Ｍリン酸ナトリウム緩衝液（ｐＨ５．５）中、４０℃で行った。酵素溶液０．１ｍｌに、０．１Ｍリン酸ナトリウム緩衝液（ｐＨ５．５）を０．２ｍｌ加え、２０ｍＭ ＯＮＰＧを０．２ｍｌ添加することで反応を開始し、１０分後に０．１Ｍの炭酸ナトリウム溶液を５ｍｌ添加することで反応を停止させた。生成したｏ−ニトロフェノールの量を吸光度４１５ｎｍで測定した。
ｏ−ニトロフェニルβ−Ｄ−ガラクトピラノシド、ｏ−ニトロフェニルβ−Ｄ−グルコピラノシドを基質とした時も同様に試験し、生成したｏ−ニトロフェノールを定量した。
【００２２】
ラフィノース、メリビオース、スクロース、ラクトース及びマルトースを基質とした時も、反応停止を沸騰水に５分間保持することで行った以外、同様に試験した。その後、ラフィノースを基質とした時は、ガラクトースＵＶテストを用いてガラクトースを定量し、メリビオース、スクロース、ラクトース及びマルトースを基質とした時は、グルコスタット法によりグルコースを定量した。
ここで、酵素１単位は、本酵素反応条件下で、１分当たりに１μmolの反応生成物を生じる酵素量とした。
【００２３】
（５）至適温度及び安定温度範囲
図４に、本酵素の温度に対する影響を示した。安定性の検討は、酵素溶液を各温度で２０分間保持した後、素早く４℃に冷却し、基質をＯＮＰα−ガラクトシドを用いて、正反応の酵素活性を定法に従い測定した。その結果、至適温度は６０℃であり、６０℃まで安定であった。図面からも明らかなように、本酵素は非常に高い耐熱性を有する点で、きわめて特徴的である。
【００２４】
（６）ｐＨ、温度等による失活の条件
特に詳細を検討していないが、（３）及び（５）に記載のｐＨ及び温度の条件、特に安定性において、相対活性が低下している範囲以上が失活の条件であると考察される。また、酵素溶液を沸騰水中に５分間保持した後の酵素活性消失は確認している。
【００２５】
（７）精製方法
本Escherichia coli BL21(DE3)-pET32Trx/galαの発現系には、プラスミドベクターpET32-Ek/LIC由来のHis・Tag配列が含まれており、無細胞抽出液からアフィニティークロマトグラフィーにより１段階で発現α−ガラクトシダーゼを精製することができる様に工夫されている。His・Tag配列は、ヒスチジン残基を６から１０個ほど並べて配した配列で、金属イオンと結合能を有している。このため発現産物を、金属イオンと結合したHis・Tag Resinによるキレートクロマトグラフィーによって高効率に単一タンパク質が精製できる。手法については公知の事実でありNovagent社の精製マニュアルに従った。その結果、大腸菌E.coli BＬ21(DE3)-pET32Trx/galαの無細胞抽出液から回収率８０％以上の高回収率で、α−ガラクトシダーゼを単一に精製した。
【００２６】
（８）分子量及び分子量の測定方法
発現α−ガラクトシダーゼタンパク質の分子量測定は、タンパク質の精製純度検定と同時に、Laemmli(Laemmli: Nature（London）、１９７０、２２７、６８０−６８５)の方法に従いＳＤＳ−ＰＡＧＥにより行った。ゲルの濃度は７．５％とした。タンパク質の染色はCoomassie Brilliant Blueを用いた。その結果、分子量は、およそ８２，０００Ｄａと測定された。この値は、遺伝子のＤＮＡ塩基配列から推定されるアミノ酸配列から算出される分子量８２，７１２Ｄａと類似しており、信頼性がある。
【００２７】
本発明に係る新規耐熱性酵素は、上記した理化学的性質を有する酵素であればすべてのものが包含され、例えば配列番号１のアミノ酸配列で示されるタンパク質もその１例として例示される。その製造方法についても、その遺伝子を含有した新規形質転換体（ＦＥＲＭ ＢＰ−７１４０として生命研に国際寄託されている）をＩＰＴＧで誘導培養したり、誘導によることなく通常培養することにより、本発明に係る新規耐熱性α−ガラクトシダーゼを得ることができるし、その新規アミノ酸配列の１例が明らかにされたので（配列番号１）、合成することによっても本酵素を得ることができる。
【００２８】
本酵素は、正反応であるα−ガラクトシド結合の分解を高効率で行い、例えばメリビオースやラフィノースを効率的に分解できるだけでなく、逆反応であるラフィノース合成を非常に効率的に実施することができる。ラフィノース合成は、スクロース、ガラクトース（及び／又はＵＤＰ−ガラクトース）の存在下、本発明に係る新規耐熱性α−ガラクトシダーゼを用いてインキュベートすることによって、非常に効率的に実施することができる。
【００２９】
ラフィノース合成は、例えば次のようにして実施することができる。例えば、本酵素としては、精製品のほか、粗製品も使用可能であることはもちろんのこと、形質転換体の培養物、培養液、無細胞抽出液、それらの濃縮物等の少なくともひとつが適宜使用可能である。スクロース、ガラクトースの基質濃度は高い方が良く、各２０％以上、好ましくは２５％以上とすると好適であるが、好適例として、スクロース４０〜８０％（好ましくは５０〜７０％）、ガラクトース５〜４５％（好ましくは１５〜３５％）が例示される。
【００３０】
反応ｐＨは、６．０よりも中性〜アルカリ側とするのが良く、反応温度は、正反応が生じないような高温とするのが良い。具体的には、５０〜７５℃以上、好ましくは６０〜７３℃以上、更に好ましくは、酵素の安定性はやや低下するけれども、反応温度を約７０℃に設定すればよい。
【００３１】
この場合、本酵素は卓越した耐熱性を有する特徴を有するため、ラフィノースの合成を効率的に行うことがはじめて可能となった点で画期的である。そのうえ、本酵素は高温においても活性を有しているため、反応系を高温に維持することができ、反応系が雑菌によって汚染されることがなく、工業的実施に特に好適である。また、酵素液、その他反応液を高温殺菌することも可能であって、本発明はこの点においても非常にすぐれている。
【００３２】
なお、反応条件としては、反応速度、収率等を考慮に入れないのであれば、上記した範囲から逸脱してもさしつかえなく、適宜選択することができる。
以下、試験例及び実施例を挙げて、本発明を更に具体的に説明する。
【００３３】
〔試験例〕
（１）８０８４株のα−ガラクトシダーゼの酵素精製
酵素の精製操作は特に断らない限り４℃で行い、緩衝液は１０ｍＭリン酸ナトリウム緩衝液、ｐＨ７．０（以後、緩衝液とする）を用いた。
【００３４】
８０８４株を本庄らの方法（本庄ら、精糖技術研究会誌、１９８５、３５、６７〜７３）に従って培養した。培養菌体を６倍量のグリシン−水酸化ナトリウム緩衝液、ｐＨ８．７に懸濁し、５０℃で２４時間自己消化を行った。その後、遠心分離により菌体残渣を除き、得られた上清液を粗酵素液とした。また、この様に培養した湿菌体は（３）でも使用した。
【００３５】
この粗酵素液をゆっくりと撹拌しながら５０％飽和になるよう硫酸アンモニウムを添加し、１時間放置した。この操作で塩析したタンパク質を遠心分離処理で沈殿物として回収した。この沈殿物を少量の緩衝液に溶解し、同緩衝液で一晩透析した。
【００３６】
この粗酵素液を担体として緩衝液で平衡化したDEAE Sephacelを用いたイオン交換カラムクロマトグラフィー（２．７×４５ｃｍ）に供した。吸着したタンパク質の溶出は緩衝液中に含まれる塩化ナトリウム濃度を０〜０．５Ｍまで直線的に変化させて行った。定法に従い、α−ガラクトシダーゼ活性画分を測定し、活性画分を遠心限外ろ過膜で濃縮した。
【００３７】
この粗酵素液を担体として緩衝液で平衡化したSephacryl S-300を用いたゲルろ過カラムクロマトグラフィー（２．５×５０ｃｍ）に供した。活性画分を遠心限外ろ過膜で濃縮した。
この粗酵素液を担体として緩衝液で平衡化したDEAE-Sepharoseイオン交換カラムクロマトグラフィー（１．８×１３ｃｍ）に供した。吸着したタンパク質の溶出は緩衝液中に含まれる塩化ナトリウムの濃度を０〜０．３Ｍまで直線的に変化させて行った。活性画分を遠心限外ろ過膜で濃縮した。
【００３８】
タンパク質の精製純度はLaemmli(Laemmli:Nature(London)、１９７０、２２７、６８０〜６８５）の方法に従いＳＤＳ−ＰＡＧＥにより検定した。ゲルの濃度は７．５％とした。タンパク質の染色はCoomassie Briliant Blueを用いた。
以上の操作で８０８４株のα−ガラクトシダーゼタンパク質を電気泳動的に単一にまで精製できた。回収率は３６％で、比活性は２１６．２Ｕ／ｍｌであった。
【００３９】
（２）精製α−ガラクトシダーゼの内部アミノ酸配列およびオリゴヌクレオチドプライマーの合成
【００４０】
（１）で調製した精製酵素標品とリジルエンドペプチダーゼとを、０．１Ｍトリス−塩酸緩衝液（ｐＨ９．０）中で混合し、３７℃で２４時間反応させてポリペプチド鎖を分解した。反応後の分解ペプチド断片をＳＤＳ−ＰＡＧＥにより分離し、電気泳動後のゲルをＰＶＤＦ膜に転写し、転写されたペプチド鎖を切り出し、それぞれのＮ−末端アミノ酸配列を気相式プロテインシーケンサーに供して解読した。
その結果、４カ所の内部アミノ酸配列を解読することができた。これらの配列は、配列番号１に全て含まれることになった（図１）。
【００４１】
決定したアミノ酸配列を基に、配列番号３、４（図５、６）に示す２種類の合成オリゴヌクレオチドプライマーを作製した。即ち、縮重の少ないアミノ酸配列を多く含む部位を選択し、すでにＤＮＡのデーターベース、DNA Information Stock Center、DISC(http://www.dna.affrc.go.jp/,National Institute of Agrobiological Resources, Tsukuba）に登録されている糸状菌アブシディア属のコドン使用頻度を考慮に入れた。また、イノシンも併用した。
【００４２】
使用したセンスプライマー、アンチセンスプライマーを配列番号３、４（図５、６）に示す。なお、配列中、ｖはＡ又はＧ、ｙはＴ又はＣ、ｎはＩ（イノシン）を示す。
【００４３】
（３）８０８４株の染色体ＤＮＡの調製
これからの実験で多用する遺伝子クローニング実験の基礎技術は、斯界において公知のものであり、特に断らない限りSambrookらの方法（Sambrook et al.：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）に従って行った。
【００４４】
（１）で調製した８０８４株の湿菌体約２ｇをＴＥＮ緩衝液（１０ｍＭトリス塩酸、１ｍＭ ＥＤＴＡ、０．１Ｍ ＮａＣｌ、ｐＨ８．０）に懸濁し、良く洗浄した。遠心分離して菌体を回収し、同洗浄操作を３回繰り返した。その後、洗浄菌体をマイナス８０℃で冷凍してから、凍結乾燥を行った。その後、乾燥菌体を乳鉢ですりつぶし、粉末状にした。この粉末にリゾチームとＮ−アセチルムラミデイスをそれぞれ添加し、３７℃で２４時間インキュベートした。処理物をマイナス８０℃で１時間凍結し、ＴＥＮ緩衝液を加え３７℃に加温し、３．３％（ｗ／ｖ）となるようにＳＤＳを加えさらに３７℃で３時間インキュベートし細胞を破砕した。この溶液をフェノール処理後、エタノール沈澱し析出した核酸をガラス棒に巻き付け回収した。この核酸を７０％のエタノールで洗浄後、乾燥し、ＴＥに再溶解しさらにＲＮａｓｅ処理、フェノール処理、フェノール−クロロフォルム処理後、エタノール沈澱し、回収し、染色体ＤＮＡとした。この操作により約１０ｍｇの染色体ＤＮＡを調製することができた。
【００４５】
（４）プローブ合成と確認
（２）で作製したプライマーと（３）で調製した染色体ＤＮＡを混合し、Ｔａｑ ＤＮＡポリメラーゼとＤｉｇ−１１−ｄＵＴＰの蛍光色素を用いた系でＰＣＲを行い（総液量は５０マイクロリットルで行い、サイクル数は３５回とし、１サイクルは９８℃−１分間、６８℃−３分間とした）、増幅された約１８０塩基対の蛍光ラベルＤＮＡ断片をプローブとした。
【００４６】
また、作製したプローブは、８０８４株から（５）に記した手法で抽出したｍＲＮＡを、ホルムアミド−ホルムアルデヒドアガロースゲル電気泳動に供し分離して、キャピラリー法でナイロンメンブランにブロットされ、ノーザンブロット解析して、実験に使用可能であることを確認した。プローブとのハイブリダイゼーション条件は、ハイブリ溶液（６×ＳＳＣ（１×ＳＳＣは０．１５Ｍ ＮａＣｌ、０．０１５Ｍクエン酸三ナトリウム）、３％ブロッキング試薬、０．１％ＳＤＳ、１０ｍＭ ＥＤＴＡ、１５０マイクログラム／ｍｌのサケ精子ＤＮＡ）中で６８℃で一晩とした。
【００４７】
（５）８０８４株のｃＤＮＡ遺伝子ライブラリーの構築
８０８４株を培養し、菌体から全ＲＮＡを抽出して、マイナス８０℃で保存した。
ポリＡ ｍＲＮＡを簡易カラムを用いて精製した後、ｃＤＮＡを合成した。合成したｃＤＮＡは、ｐＳＰＯＲＴ IIプラスミドベクターのマルチクローニング部位に挿入した。このプラスミドベクター群を、大腸菌５α株にエレクトロポレーション法で形質転換してｃＤＮＡライブラリーを構築した。形質転換体は１５０マイクログラム／ｍｌのアンピシリンを含むＬＢ寒天培地（１０ｇ／Ｌポリペプトン、５ｇ／Ｌ酵母エキス、１０ｇ／Ｌ ＮａＣｌ、１５ｇ／Ｌ寒天、ｐＨ７．０）に出現するコロニーをもって確認した。
【００４８】
（６）８０８４株のα−ガラクトシダーゼ遺伝子の取得およびその一次配列
（５）で作製した遺伝子ライブラリーの大腸菌コロニーのＤＮＡをナイロンメンブランにブロットした。（４）にて作製したＤｉｇラベル化プローブを用いて、（４）のハイブリダイゼーションと同じ条件で陽性クローンをコロニーハイブリダイゼーションでスクリーニングした。スクリーニングは２段階選抜を行い、最終選別として大腸菌を単一コロニー化した後、プラスミドＤＮＡを抽出しフラグメントサザンハイブリダイゼーションを行って強いシグナルが確認できるものを陽性クローンとした。
【００４９】
目的遺伝子を含む可能性がある領域のＤＮＡ断片を鋭意サブクローニングし、ＤＮＡの塩基配列を決定した。一連の操作は宝酒造（株）遺伝子解析センターに依託した。
【００５０】
ＤＮＡシーケンスの解析はＧＥＮＥＴＹＸ−ＭＡＣを用いた。ＤＮＡシーケンスに関する様々な情報はDNA Information Stock Center、ＤＩＳＣのネットワークサービスを利用した。
【００５１】
その結果、配列番号２（図２）に示す５′末端からのＤＮＡ塩基配列を有する２，１９０塩基対のα−ガラクトシダーゼの構造遺伝子を解読した。この配列はこれまで見い出されていない新規な遺伝子であった。また、このＤＮＡ塩基配列より類推される８０８４株が生産するα−ガラクトシダーゼは７２９個のアミノ酸からなり、配列番号１（図１）に示すようなＮ末端からのアミノ酸配列を有していた。
【００５２】
（７）α−グルコシダーゼ遺伝子の発現プラスミドベクターの構築及び形質転換
クローン化されたα−ガラクトシダーゼＤＮＡ断片を、構造遺伝子が完全な形で取り出せる制限酵素（ＥｃｏＲＩとＮｏｔＩ）で切断し、平滑末端化し、切断部位間の約２．２キロ塩基対の領域をｐＥＴ３２−ＢＫ／ＬＩＣプラスミドベクターのthioredoxin配列の下流に連結し、新たなプラスミドベクターｐＥＴ３２Ｔｒｘ／ｇａｌαを構築した。このプラスミドベクターには大腸菌中で外来遺伝子として連結された遺伝子を効率的に転写、翻訳できるＴ７Ｌａｃプロモーターが導入されており（８）に示す培養方法でα−ガラクトシダーゼを高効率で発現・製造させることができる。
【００５３】
この遺伝子組換えプラスミドベクターを大腸菌ＢＬ２１（ＤＢ３）株のコンピテント細胞にヒートショック法で形質転換し、組換え微生物を創製した。この微生物はEscherichia coli BL21(DE3)-pET32Trx/galαと命名し、工業技術院生命工学工業技術研究所に、ＦＥＲＭ ＢＰ−７１４０として国際寄託した。
【００５４】
（８）形質転換体の培養及びα−ガラクトシダーゼの発現
（７）で作製した大腸菌BL21(DE3)-pET32Trx/galαを１５０マイクログラム／ｍｌのアンピシリンを含む５ｍｌのＬＢ培地で３７℃で対数増殖中期まで培養した。この菌液を終濃度で１ｍＭとなるようにイソプロピル−β−Ｄ−チオガラクトピラノシド（以後、ＩＰＴＧと略する）を含む１００ｍｌの同培地に接種し、３７℃で振とう培養した。培養後、大腸菌を遠心分離して回収した。菌体は１０ｍＭリン酸ナトリウム緩衝液（ｐＨ６．０）で２回洗浄後、同緩衝液に再懸濁し、超音波破砕機で破砕した。破砕液を遠心分離し、この上清を無細胞抽出液とした。
【００５５】
この様に調製した大腸菌BL21(DE3)-pET32Trx/galαの無細胞抽出液のα−ガラクトシダーゼ活性を定法に従い測定したところ、１８．７単位／培養液（ｍｌ）であった。８０８４株は最適条件下でも０．４４９単位／培養液（ｍｌ）のα−ガラクトシダーゼしか生産できないため、本発明により遺伝子組換え体は、元株８０８４より高い生産性を獲得した。活性の比較を表２に示した。
【００５６】

【００５７】
また、この無細胞抽出液から発現α−ガラクトシダーゼは、プラスミドベクター由来のヒスチジンＴａｇを利用したアフィニティークロマトグラフィーで１回の操作で、電気泳動的に完全精製することができ、回収率９２％で、完全にかつ安価に発現α−ガラクトシダーゼが供給できる。
【００５８】
【実施例】
（発現α−ガラクトシダーゼを用いたラフィノースの製造）
スクロースが終濃度で６００ｇ／Ｌ、ガラクトースが終濃度で２５０ｇ／Ｌとなるように、１０ｍＭリン酸緩衝液（ｐＨ７．０）に溶解した液を基質溶液とした。この基質溶液と（８）で調製した精製遺伝子組換え発現α−ガラクトシダーゼ溶液（１００Ｕ分）とを混合し、７０℃で酵素反応を行った。反応終了後、沸騰水中で５分間保持することで反応を停止した。この反応で合成されたラフィノースはＨＰＬＣを用いた系で測定した。カラムは、Ｓｈｏｄｅｘ ＳＵＧＡＲ ＫＳ８０１（８．０×３００ｍｍ）、溶離液は水、検出器はＲＩを用いた。
【００５９】
その結果、表３に示すように、ラフィノースの収率は使用したガラクトースの１０％であった。つまり、０．１４モル／Ｌ（２５ｇ／Ｌ）のラフィノースが合成された。また、ガラクトースをＵＤＰ−ガラクトースに変更して基質として用いた時、ラフィノースの収率は４５％と増加した。つまり、０．６３モル／Ｌ（１１３ｇ／Ｌ）のラフィノースが合成された。
【００６０】

【００６１】
なお、本実施例においては、上記のように新規形質転換体Escherichia coli BL21(DE-3)-pET32Trx/galαをＩＰＴＧで誘導培養し、無細胞抽出液を調製した。本培養条件で、１８．７単位／培養液ｍｌのα−ガラクトシダーゼが生産できる。本酵素をアフィニティークロマトグラフィーで精製することができるが、工業的なラフィノースの生産を考慮した場合、コスト面に関わってくるので、ラフィノースの生産効率の面で何の問題もないと思われるため、無細胞抽出液をそのまま使用する事とした。
【００６２】
反応液の全量を５００ｍｌとして、スクロースが終濃度で６００ｇ／Ｌ、ガラクトースが終濃度で２５０ｇ／Ｌとなるように、１０ｍＭリン酸緩衝液（ｐＨ７．０）に溶解した液を基質溶液とした。この基質溶液と調製したα−ガラクトシダーゼ溶液（１００単位分）とを混合し、７０℃で酵素反応を行った。反応終了後、沸騰水中で５分間保持することで反応を停止した。
【００６３】
本酵素の正反応での至適ｐＨは５．５であったが、逆反応のラフィノース合成反応では７．０であったため、正反応が起こりにくくするため、１０ｍＭリン酸緩衝液のｐＨを７．０と設定した。
【００６４】
また、ラフィノース合成反応は、できる限り高温で行う方が高効率なため（通常の４０℃付近の反応温度では、正反応が行われてしまう）、酵素の安定性は悪化するが、反応温度を７０℃に設定した。
【００６５】
また、スクロース、ガラクトースの基質濃度は、各２５％以上でないとラフィノース合成反応が進行しなかった。そのため、酵素の安定性は悪化するが、スクロース６０％、ガラクトース２５％と設定した。
【００６６】
【発明の効果】
本発明のラフィノースの新規製造方法は、耐熱性α−ガラクトシダーゼを用いることによってはじめて可能となったものである。本酵素は、ガラクトシド結合を効率的に分解する作用を有するだけでなく、その逆反応を利用して、スクロースとガラクトースからラフィノースを大量合成することが可能となった。この逆反応は高温条件下で実施する必要があるため、反応中雑菌による汚染が防止されており、きわめて優れた工業的ラフィノースの製造方法ということができる。
【００６７】
【配列表】

【図面の簡単な説明】
【図１】８０８４株由来の耐熱性α−ガラクトシダーゼのアミノ酸配列を示す。
【図２】８０８４株由来の耐熱性α−ガラクトシダーゼ遺伝子ＤＮＡの塩基配列を示す。
【図３】発現α−ガラクトシダーゼのｐＨに対する影響を示す。
【図４】発現α−ガラクトシダーゼの温度に対する影響を示す。
【図５】センスプライマーの塩基配列を示す。
【図６】アンチセンスプライマーの塩基配列を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing raffinose, and more particularly to a novel method for producing raffinose using a novel thermostable α-galactosidase.
[0002]
In addition, the newly analyzed α-galactosidase gene of Absidia corymbifera IFO 8084 (hereinafter sometimes referred to as 8084 strain) is novel, and this structural gene is linked to an expression plasmid vector. By introducing it into a system using Escherichia coli as a host, α-galactosidase can be efficiently expressed in large quantities and can be supplied in large quantities at a low cost to the industrial field using this enzyme. A novel thermostable α-galactosidase gene obtained from the filamentous fungus Absidia corymbifera according to the present invention, a recombinant plasmid obtained by linking this structural gene to an expression plasmid vector, and introduced into a host microorganism All of the transformants are conventionally unknown.
[0003]
Here, since the industrial production of a novel thermostable α-galactosidase has become possible, the raffinose industry, which uses sucrose and galactose and / or UDP-galactose as raw materials for raffinose, which has been extremely difficult to produce industrially in the past. Manufacturing is possible, and the present invention has been established.
[0004]
[Prior art]
In recent years, with the diversification of eating habits, lifestyle-related diseases and the like have been regarded as serious problems, and consumers' awareness of foods and food materials has increased. Under such circumstances, excessive intake of sugar has been taken up, and the development and production of oligosaccharides as new sweeteners with low calories and physiological functions have become important, and the research is also diverse. .
[0005]
The inventors focused on Raffinose from such a viewpoint. Raffinose is a trisaccharide oligosaccharide having a structure in which D-galactose is linked to α1-6 in a sucrose molecule. In the natural world, beet (sugar radish), a raw material for sugar, seeds of legumes such as soybeans, sugar cane, honey, cabbage, yeast, potatoes, grapes, wheat and corn are widely distributed. Of course, sweeteners are used, but as a stabilizer for freezing and storing bovine semen, it is also blended in transport solutions for transplantation of human organs used in various countries around the world. On the other hand, as research on human intestinal bacteria progresses, indigestible oligosaccharides have attracted attention as a factor that causes growth of bifidobacteria, which are useful in the intestines. As a result of clinical research, raffinose has been found to be bifidobacteria. It became clear that it had an excellent proliferation effect, and was approved as a food material for specified health use in 1993 by the Ministry of Health and Welfare. In addition, medical clinical research has revealed that oral administration of raffinose is effective for atopic dermatitis, which is one of the pathologies of allergies. Yes.
[0006]
Raffinose is industrially recovered as a by-product during the production of beet sugar. Raffinose in the beet is considered to be a bioprotective component against freezing because it increases with decreasing temperatures before and after autumn harvest. However, the maximum raffinose content in the beet is only about 0.1% of the rhizome weight, and the production amount is greatly related to the sugar production amount, but there is a limit. In the future, as described above, the physiological function of raffinose will become clear, and an increase in demand will be essential, and not only extraction production from plants but also synthetic products from inexpensive raw materials will be required.
[0007]
Prior to the present invention, the inventors used an α-galactosidase activity of 8084 strain for the purpose of degrading raffinose, which was a component that hinders the crystallization of sugar in the sugar production process, and conducted an efficient method for degrading raffinose. Established. It is theoretically possible to synthesize raffinose using sucrose and galactose as raw materials for this α-galactosidase as a reverse reaction.
[0008]
[Problems to be solved by the invention]
As described above, α-galactosidase is not only used in the sugar industry etc. for the purpose of decomposing raffinose which interferes with crystallization of sugar by utilizing the original property of decomposing α-galactoside, but also the reverse reaction thereof. Accordingly, it can be used for the production of raffinose using sucrose and galactose as raw materials.
[0009]
In the sugar industry, a large amount of α-galactosidase is required for degrading raffinose, and a large amount of α-galactosidase is also required for the synthesis of raffinose utilizing the reverse reaction. In other words, as mentioned earlier, the production of raffinose using this enzyme uses the reverse reaction instead of the original normal reaction, so that the efficiency is extremely deteriorated when the enzyme is in a small amount or the reaction conditions are not optimal. there is a possibility. In the present invention, since a sufficient amount of α-galactosidase is supplied to the method for producing raffinose, efficient enzyme production was detected at the gene level and succeeded. The purpose of the present invention was to introduce a genetic engineering technique to mass-produce enzymes, and the present invention was achieved.
[0010]
[Means for Solving the Problems]
The present invention has been made in order to achieve the above-described object, and the object microorganisms and enzymes have been created by genetic engineering techniques.
[0011]
Therefore, the inventors cloned the α-galactosidase gene from the chromosomal DNA of the 8084 strain, elucidated the primary sequence of the structural gene, ligated the DNA fragment containing this to an expression plasmid vector, and Escherichia coli (Escherichia coli). ) BL21 (DE3) strain (hereinafter sometimes referred to as E. coli) was transformed, the cells were cultured, and a large amount of α-galactosidase was expressed. The obtained α-galactosidase has not only excellent α-galactoside resolution, but also can efficiently synthesize raffinose by the reverse reaction, and also has activity at a very high temperature of 70 ° C. The enzyme was identified as a novel enzyme that was previously unknown, after confirming that it had such characteristics as extremely high heat resistance.
Hereinafter, the present invention will be described in detail.
[0012]
In order to clone the 8084 strain α-galactosidase gene, the internal amino acid sequence of the enzyme-purified α-galactosidase protein was first determined. The 8084 strain was cultured, and expressed α-galactosidase was electrophoretically purified to single using ammonium sulfate fractionation and various column chromatography. The purified α-galactosidase protein was cleaved with a proteolytic enzyme, the resulting peptide chain group was separated, and each N-terminal amino acid sequence was decoded by a peptide sequencer.
An oligonucleotide primer was synthesized based on the amino acid sequence thus obtained.
[0013]
Separately, chromosomal DNA of 8084 strain prepared separately was used as a template, mixed with oligo DNA previously synthesized as a primer, and the α-galactosidase gene was partially amplified by PCR method, and this was used as a probe.
[0014]
Then, a chromosome was extracted from the 8084 strain, cDNA was synthesized from the obtained chromosome, the synthesized cDNA was inserted into a multicloning site of a plasmid vector, and a host such as E. coli was transformed using this plasmid vector group, A cDNA library was constructed. From the E. coli colonies of the gene library thus prepared, screening was performed by colony hybridization to select positive clones. Plasmid DNA was extracted from the positive clonal strain, and the DNA base sequence of the region possibly containing the target α-galactosidase gene was decoded. As a result, the total DNA base sequence of the structural gene of the α-galactosidase gene was determined. This base sequence is shown in SEQ ID NO: 2 in the sequence listing (FIG. 2), and this sequence was a novel gene that was not conventionally known. Moreover, from this DNA base sequence, the amino acid sequence of α-galactosidase derived from 8084 strain is inferred to be SEQ ID NO: 1 (FIG. 1).
[0015]
Then, a fragment completely containing the structural gene of α-galactosidase is inserted and ligated downstream of the thioredoxin sequence of an E. coli expression plasmid vector such as pET32-EK / LIC plasmid vector (Takara Shuzo Co., Ltd.), and a new recombinant plasmid Built. In this plasmid vector, a promoter capable of efficiently expressing a gene linked as a foreign gene in E. coli is introduced, and this recombinant plasmid is transformed into E. coli and cultured in induction to express a large amount of α-galactosidase. Made possible.
[0016]
The α-galactosidase according to the present invention has the following physicochemical properties, not only has a high α-galactoside resolution as a normal reaction, but also has a property of synthesizing raffinose from sucrose and galactose as a reverse reaction, It exhibits excellent characteristics such as an optimum temperature range of 50 to 70 ° C. and extremely high resistance to heat. Such α-galactosidase is a novel enzyme that has not been found in the past and is unknown in the past.
[0017]
(1) Action
This enzyme generally performs the reaction as defined by α-galactosidase (cleaves the α-galactoside bond at the non-reducing end of the sugar chain and has exoglycosidase activity) as a positive reaction, and particularly described in (2) Has substrate specificity.
As a reverse reaction, a reaction for synthesizing raffinose is performed using sucrose and galactose as substrates. The test method for substrate specificity and the test method for enzyme titer are described in (4), and detailed reaction conditions for the reverse reaction are described later.
[0018]
(2) Substrate specificity
Table 1 shows the relative activity of each substrate when the activity of the enzyme with respect to ONPα-galactoside is taken as 100% (positive substrate specificity). As a result, this enzyme had very high substrate specificity for ONPα-galactoside.
[0019]

[0020]
(3) Optimum pH and stable pH range
FIG. 3 shows the effect of this enzyme on the pH. The stability study was carried out by holding the enzyme solution in each pH buffer solution for 24 hours at 4 ° C., then replacing the solution with a pH 5.5 buffer solution, and using ONPα-galactoside as a substrate for positive reaction. The enzyme activity was measured according to a standard method. As a result, the optimum pH of this enzyme was 5.5, and it was stable at pH 5.0 to 11.0.
[0021]
(4) Measuring method of titer
This enzyme activity was measured according to the ONPG (o-nitrophenyl α-D-galactopyranoside) method. The enzyme reaction was performed at 40 ° C. in 0.1 M sodium phosphate buffer (pH 5.5). The reaction was started by adding 0.2 ml of 0.1 M sodium phosphate buffer (pH 5.5) to 0.1 ml of the enzyme solution, and adding 0.2 ml of 20 mM ONPG. After 10 minutes, 0.1 M sodium carbonate was added. The reaction was stopped by adding 5 ml of the solution. The amount of o-nitrophenol produced was measured at an absorbance of 415 nm.
The same test was performed when o-nitrophenyl β-D-galactopyranoside and o-nitrophenyl β-D-glucopyranoside were used as substrates, and the produced o-nitrophenol was quantified.
[0022]
When raffinose, melibiose, sucrose, lactose and maltose were used as substrates, the same test was conducted except that the reaction was stopped by maintaining in boiling water for 5 minutes. Thereafter, when raffinose was used as a substrate, galactose was quantified using a galactose UV test, and when melibiose, sucrose, lactose and maltose were used as substrates, glucose was quantified by a glucostat method.
Here, 1 unit of enzyme was defined as the amount of enzyme that produced 1 μmol of reaction product per minute under the enzyme reaction conditions.
[0023]
(5) Optimum temperature and stable temperature range
FIG. 4 shows the effect of this enzyme on temperature. The stability was examined by holding the enzyme solution at each temperature for 20 minutes, then quickly cooling to 4 ° C., and using the substrate ONPα-galactoside, the enzyme activity of the positive reaction was measured according to a standard method. As a result, the optimum temperature was 60 ° C. and was stable up to 60 ° C. As is apparent from the drawings, this enzyme is very characteristic in that it has a very high thermostability.
[0024]
(6) Conditions for deactivation due to pH, temperature, etc.
Although the details are not examined, it is considered that the conditions for deactivation are above the range where the relative activity is reduced in the conditions of pH and temperature described in (3) and (5), particularly the stability. . Moreover, the enzyme activity loss | disappearance after hold | maintaining an enzyme solution in boiling water for 5 minutes has been confirmed.
[0025]
(7) Purification method
This Escherichia coli BL21 (DE3) -pET32Trx / galα expression system contains a His • Tag sequence derived from the plasmid vector pET32-Ek / LIC and is expressed in one step from the cell-free extract by affinity chromatography. -It has been devised to be able to purify galactosidase. The His • Tag sequence is a sequence in which about 6 to 10 histidine residues are arranged side by side and has a binding ability to metal ions. For this reason, the expression product can be purified with high efficiency by chelate chromatography using His • Tag Resin coupled with metal ions. The method is a known fact and follows the purification manual of Novagent. As a result, α-galactosidase was purified from a cell-free extract of E. coli BL21 (DE3) -pET32Trx / galα with a high recovery rate of 80% or more.
[0026]
(8) Molecular weight and molecular weight measurement method
The molecular weight of the expressed α-galactosidase protein was determined by SDS-PAGE according to the method of Laemmli (Laemmli: Nature (London), 1970, 227, 680-685) simultaneously with the protein purification purity test. The gel concentration was 7.5%. Coomassie Brilliant Blue was used for protein staining. As a result, the molecular weight was measured to be approximately 82,000 Da. This value is similar to the molecular weight 82,712 Da calculated from the amino acid sequence deduced from the DNA base sequence of the gene and is reliable.
[0027]
The novel thermostable enzyme according to the present invention includes all enzymes having the above-mentioned physicochemical properties. For example, the protein represented by the amino acid sequence of SEQ ID NO: 1 is exemplified as an example. As for the production method, a novel transformant containing the gene (deposited internationally at Life Research Institute as FERM BP-7140) is induced and cultured with IPTG or is usually cultured without induction. Thus, the novel thermostable α-galactosidase can be obtained, and since one example of the novel amino acid sequence has been clarified (SEQ ID NO: 1), the enzyme can also be obtained by synthesis.
[0028]
This enzyme can efficiently decompose the α-galactoside bond, which is a normal reaction, and can efficiently decompose, for example, melibiose and raffinose, as well as the reverse reaction of raffinose. . Raffinose synthesis can be carried out very efficiently by incubating with the novel thermostable α-galactosidase according to the present invention in the presence of sucrose, galactose (and / or UDP-galactose).
[0029]
Raffinose synthesis can be carried out, for example, as follows. For example, as the enzyme, not only a purified product but also a crude product can be used, and at least one of a transformant culture, a culture solution, a cell-free extract, and a concentrate thereof is appropriately used. It can be used. The substrate concentration of sucrose and galactose is preferably high, and each is preferably 20% or more, and preferably 25% or more. Preferred examples include sucrose 40 to 80% (preferably 50 to 70%), galactose 5 45% (preferably 15 to 35%) is exemplified.
[0030]
The reaction pH should be neutral to alkali side than 6.0, and the reaction temperature should be high so that no positive reaction occurs. Specifically, the reaction temperature may be set to about 70 ° C., although the enzyme stability is slightly reduced, although it is 50 to 75 ° C. or higher, preferably 60 to 73 ° C. or higher.
[0031]
In this case, the present enzyme has an excellent thermostability characteristic, and thus is revolutionary in that it is possible for the first time to efficiently synthesize raffinose. In addition, since the present enzyme is active even at high temperatures, the reaction system can be maintained at high temperatures, and the reaction system is not contaminated by various bacteria, which is particularly suitable for industrial implementation. In addition, the enzyme solution and other reaction solutions can be sterilized at a high temperature, and the present invention is also excellent in this respect.
[0032]
The reaction conditions can be selected as appropriate without departing from the above-mentioned range if the reaction rate, yield, etc. are not taken into consideration.
Hereinafter, the present invention will be described more specifically with reference to test examples and examples.
[0033]
[Test example]
(1) Enzymatic purification of 8084 strain α-galactosidase
The enzyme purification procedure was performed at 4 ° C. unless otherwise specified, and a 10 mM sodium phosphate buffer, pH 7.0 (hereinafter referred to as a buffer) was used as the buffer.
[0034]
The 8084 strain was cultured according to the method of Honjo et al. (Honjo et al., Journal of Refined Sugar Technology, 1985, 35, 67-73). The cultured cells were suspended in 6 times the amount of glycine-sodium hydroxide buffer, pH 8.7, and self-digested at 50 ° C. for 24 hours. Thereafter, the cell residue was removed by centrifugation, and the obtained supernatant was used as a crude enzyme solution. The wet cells cultured in this way were also used in (3).
[0035]
While slowly stirring this crude enzyme solution, ammonium sulfate was added to 50% saturation and left for 1 hour. The protein salted out by this operation was recovered as a precipitate by centrifugation. This precipitate was dissolved in a small amount of buffer and dialyzed overnight with the same buffer.
[0036]
The crude enzyme solution was used as a carrier and subjected to ion exchange column chromatography (2.7 × 45 cm) using DEAE Sephacel equilibrated with a buffer solution. Elution of the adsorbed protein was performed by linearly changing the concentration of sodium chloride contained in the buffer from 0 to 0.5M. According to a conventional method, the α-galactosidase active fraction was measured, and the active fraction was concentrated with a centrifugal ultrafiltration membrane.
[0037]
This crude enzyme solution was subjected to gel filtration column chromatography (2.5 × 50 cm) using Sephacryl S-300 equilibrated with a buffer solution as a carrier. The active fraction was concentrated with a centrifugal ultrafiltration membrane.
This crude enzyme solution was subjected to DEAE-Sepharose ion exchange column chromatography (1.8 × 13 cm) equilibrated with a buffer solution as a carrier. Elution of the adsorbed protein was performed by linearly changing the concentration of sodium chloride contained in the buffer from 0 to 0.3M. The active fraction was concentrated with a centrifugal ultrafiltration membrane.
[0038]
The purity of the protein is Laemmli (Laemmli: Nature (London), 1970, 227 , 680-685), and assayed by SDS-PAGE. The gel concentration was 7.5%. Coomassie Briliant Blue was used for protein staining.
Through the above operation, 8084 α-galactosidase protein was electrophoretically purified to single. The recovery rate was 36% and the specific activity was 216.2 U / ml.
[0039]
(2) Internal amino acid sequence of purified α-galactosidase and synthesis of oligonucleotide primer
[0040]
The purified enzyme preparation prepared in (1) and lysyl endopeptidase were mixed in 0.1 M Tris-HCl buffer (pH 9.0) and reacted at 37 ° C. for 24 hours to decompose the polypeptide chain. The degraded peptide fragments after the reaction are separated by SDS-PAGE, the gel after electrophoresis is transferred to a PVDF membrane, the transferred peptide chain is cut out, and each N-terminal amino acid sequence is subjected to a gas phase protein sequencer. Deciphered.
As a result, it was possible to decipher the four internal amino acid sequences. These sequences were all included in SEQ ID NO: 1 (FIG. 1).
[0041]
Based on the determined amino acid sequence, two types of synthetic oligonucleotide primers shown in SEQ ID NOs: 3 and 4 (FIGS. 5 and 6) were prepared. That is, a site containing many amino acid sequences with low degeneracy is selected, and the DNA database, DNA Information Stock Center, DISC (http://www.dna.affrc.go.jp/, National Institute of Agrobiological Resources, The frequency of codon usage of the fungus Absidia genus registered in Tsukuba) was taken into account. Inosine was also used in combination.
[0042]
The sense primer and antisense primer used are shown in SEQ ID NOs: 3 and 4 (FIGS. 5 and 6). In the sequences, v represents A or G, y represents T or C, and n represents I (inosine).
[0043]
(3) Preparation of chromosome DNA of 8084 strain
Basic techniques of gene cloning experiments to be frequently used in future experiments are known in the art, and unless otherwise specified, the method of Sambrook et al. (Sambrook et al .: Molecular Cloning a Laboratory Manual, 2nd edition, (1989).
[0044]
About 2 g of wet cells of 8084 strain prepared in (1) were suspended in TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1 M NaCl, pH 8.0) and washed well. Centrifugation collect | recovered the microbial cells, The same washing | cleaning operation was repeated 3 times. Thereafter, the washed cells were frozen at minus 80 ° C. and then freeze-dried. Thereafter, the dried cells were ground in a mortar and powdered. Lysozyme and N-acetylmuramidis were added to this powder, respectively, and incubated at 37 ° C. for 24 hours. The treated product was frozen at −80 ° C. for 1 hour, TEN buffer was added and heated to 37 ° C., SDS was added to 3.3% (w / v), and the cells were further incubated at 37 ° C. for 3 hours. It was crushed. This solution was treated with phenol, ethanol precipitated, and the precipitated nucleic acid was wound around a glass rod and collected. This nucleic acid was washed with 70% ethanol, dried, redissolved in TE, further treated with RNase, phenol, and phenol-chloroform, precipitated with ethanol, recovered, and used as chromosomal DNA. By this operation, about 10 mg of chromosomal DNA could be prepared.
[0045]
(4) Probe synthesis and confirmation
Mix the primer prepared in (2) and the chromosomal DNA prepared in (3), Taq PCR was performed in a system using DNA polymerase and Dig-11-dUTP fluorescent dye (total liquid volume was 50 microliters, the number of cycles was 35, and one cycle was 98 ° C.-1 min, 68 ° C.-3 The amplified fluorescently labeled DNA fragment of about 180 base pairs was used as a probe.
[0046]
In addition, the prepared probe was subjected to formamide-formaldehyde agarose gel electrophoresis after separating the mRNA extracted from the 8084 strain by the method described in (5), blotted on a nylon membrane by capillary method, and subjected to Northern blot analysis. , Confirmed that it can be used for experiments. Hybridization conditions with the probe were as follows: hybrid solution (6 × SSC (1 × SSC is 0.15 M NaCl, 0.015 M trisodium citrate), 3% blocking reagent, 0.1% SDS, 10 mM EDTA, 150 micrograms. / Ml salmon sperm DNA) at 68 ° C. overnight.
[0047]
(5) Construction of cDNA library of 8084 strain
The 8084 strain was cultured, and total RNA was extracted from the cells and stored at minus 80 ° C.
Poly A mRNA was purified using a simple column, and then cDNA was synthesized. The synthesized cDNA was inserted into the multiple cloning site of the pSPORT II plasmid vector. This plasmid vector group was transformed into E. coli 5α strain by electroporation to construct a cDNA library. The transformant was confirmed by colonies appearing in LB agar medium (10 g / L polypeptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, pH 7.0) containing 150 microgram / ml ampicillin.
[0048]
(6) Acquisition of α-galactosidase gene of 8084 strain and its primary sequence
The DNA of the E. coli colonies of the gene library prepared in (5) was blotted onto a nylon membrane. Using the Dig-labeled probe prepared in (4), positive clones were screened by colony hybridization under the same conditions as in (4). Screening was performed in two stages, and after colonizing E. coli as a final selection, plasmid DNA was extracted and fragment Southern hybridization was performed to confirm a strong signal as a positive clone.
[0049]
A DNA fragment in a region possibly containing the target gene was eagerly subcloned, and the base sequence of the DNA was determined. The series of operations was entrusted to the Takara Shuzo Co., Ltd. Gene Analysis Center.
[0050]
GENETYX-MAC was used for DNA sequence analysis. Various information on DNA sequences was obtained from the DNA Information Stock Center and DISC network services.
[0051]
As a result, the 2,190 base pair α-galactosidase structural gene having a DNA base sequence from the 5 ′ end shown in SEQ ID NO: 2 (FIG. 2) was decoded. This sequence was a novel gene that has not been found so far. Further, α-galactosidase produced by 8084 strain inferred from this DNA base sequence was composed of 729 amino acids and had an amino acid sequence from the N-terminus as shown in SEQ ID NO: 1 (FIG. 1).
[0052]
(7) Construction and transformation of expression plasmid vector of α-glucosidase gene
The cloned α-galactosidase DNA fragment is cleaved with restriction enzymes (EcoRI and NotI) from which the structural gene can be removed in its entirety, blunt-ended, and a region of about 2.2 kilobase pairs between the cleavage sites is pET32- A new plasmid vector pET32Trx / galα was constructed by ligation downstream of the thioredoxin sequence of the BK / LIC plasmid vector. This plasmid vector has a T7Lac promoter capable of efficiently transcribing and translating a gene linked as a foreign gene in E. coli, so that α-galactosidase can be expressed and produced with high efficiency by the culture method shown in (8). Can do.
[0053]
This recombinant plasmid vector was transformed into competent cells of E. coli BL21 (DB3) strain by the heat shock method to create a recombinant microorganism. This microorganism was named Escherichia coli BL21 (DE3) -pET32Trx / galα and internationally deposited as FERM BP-7140 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.
[0054]
(8) Culture of transformant and expression of α-galactosidase
E. coli BL21 (DE3) -pET32Trx / galα prepared in (7) was cultured in 5 ml of LB medium containing 150 microgram / ml ampicillin at 37 ° C. until the middle of logarithmic growth. This bacterial solution was inoculated into 100 ml of the same medium containing isopropyl-β-D-thiogalactopyranoside (hereinafter abbreviated as IPTG) to a final concentration of 1 mM, and cultured with shaking at 37 ° C. After cultivation, E. coli was collected by centrifugation. The cells were washed twice with 10 mM sodium phosphate buffer (pH 6.0), resuspended in the same buffer, and crushed with an ultrasonic crusher. The disrupted solution was centrifuged, and this supernatant was used as a cell-free extract.
[0055]
The α-galactosidase activity of the cell-free extract of Escherichia coli BL21 (DE3) -pET32Trx / galα prepared as described above was measured according to a conventional method, and found to be 18.7 units / culture solution (ml). Since the 8084 strain can produce only 0.449 units / ml of galactosidase even under optimum conditions, the recombinant plant according to the present invention has gained higher productivity than the original strain 8084. The activity comparison is shown in Table 2.
[0056]

[0057]
In addition, the α-galactosidase expressed from this cell-free extract can be completely purified electrophoretically in one operation by affinity chromatography using histidine Tag derived from a plasmid vector, and the recovery rate is 92%. The expressed α-galactosidase can be supplied completely and inexpensively.
[0058]
【Example】
(Production of raffinose using expressed α-galactosidase)
A solution dissolved in 10 mM phosphate buffer (pH 7.0) was used as a substrate solution so that sucrose had a final concentration of 600 g / L and galactose had a final concentration of 250 g / L. This substrate solution was mixed with the purified gene recombinant expression α-galactosidase solution (100 U) prepared in (8), and an enzyme reaction was performed at 70 ° C. After completion of the reaction, the reaction was stopped by holding it in boiling water for 5 minutes. The raffinose synthesized by this reaction was measured by a system using HPLC. The column was Shodex SUGAR KS801 (8.0 × 300 mm), the eluent was water, and the detector was RI.
[0059]
As a result, as shown in Table 3, the yield of raffinose was 10% of the galactose used. That is, 0.14 mol / L (25 g / L) raffinose was synthesized. Moreover, when the galactose was changed to UDP-galactose and used as a substrate, the yield of raffinose increased to 45%. That is, 0.63 mol / L (113 g / L) raffinose was synthesized.
[0060]

[0061]
In this example, the novel transformant Escherichia coli BL21 (DE-3) -pET32Trx / galα was induced and cultured with IPTG as described above to prepare a cell-free extract. Under the main culture conditions, 18.7 units / ml of culture broth α-galactosidase can be produced. Although this enzyme can be purified by affinity chromatography, considering the industrial production of raffinose, it is related to the cost, so there seems to be no problem in terms of raffinose production efficiency. The cell-free extract was used as it was.
[0062]
The total volume of the reaction solution was 500 ml, and a solution dissolved in 10 mM phosphate buffer (pH 7.0) was used as a substrate solution so that sucrose had a final concentration of 600 g / L and galactose had a final concentration of 250 g / L. This substrate solution and the prepared α-galactosidase solution (for 100 units) were mixed, and an enzyme reaction was carried out at 70 ° C. After completion of the reaction, the reaction was stopped by holding it in boiling water for 5 minutes.
[0063]
The optimum pH of the enzyme in the forward reaction was 5.5, but 7.0 in the reverse reaction raffinose synthesis reaction. Therefore, the pH of the 10 mM phosphate buffer was adjusted to 7 to prevent the forward reaction from occurring. Set to .0.
[0064]
In addition, the raffinose synthesis reaction is more efficient when it is performed at as high a temperature as possible (a normal reaction is performed at a reaction temperature around 40 ° C.). Set to 70 ° C.
[0065]
The raffinose synthesis reaction did not proceed unless the substrate concentrations of sucrose and galactose were 25% or more, respectively. Therefore, although the stability of the enzyme deteriorated, it was set to 60% sucrose and 25% galactose.
[0066]
【The invention's effect】
The novel method for producing raffinose of the present invention has been made possible only by using thermostable α-galactosidase. This enzyme not only has an effect of efficiently decomposing galactoside bonds, but also makes it possible to synthesize raffinose in large quantities from sucrose and galactose using the reverse reaction. Since it is necessary to carry out this reverse reaction under high temperature conditions, contamination by various bacteria during the reaction is prevented, and it can be said that this is an extremely excellent industrial method for producing raffinose.
[0067]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the amino acid sequence of thermostable α-galactosidase derived from 8084 strain.
FIG. 2 shows the base sequence of thermostable α-galactosidase gene DNA derived from 8084 strain.
FIG. 3 shows the effect of expressed α-galactosidase on pH.
FIG. 4 shows the effect of expressed α-galactosidase on temperature.
FIG. 5 shows the base sequence of the sense primer.
FIG. 6 shows the nucleotide sequence of an antisense primer.

Claims (6)

  A method for producing raffinose, comprising producing raffinose using a thermostable α-galactosidase represented by the amino acid sequence of SEQ ID NO: 1 and using sucrose and galactose and / or UDP-galactose as a substrate.   A method for producing raffinose, characterized in that raffinose is produced using α-galactosidase corresponding to the thermostable α-galactosidase gene represented by the nucleotide sequence of SEQ ID NO: 2 and using sucrose and galactose and / or UDP-galactose as a substrate. .   The method for producing raffinose according to claim 1 or 2, wherein a heat-resistant α-galactosidase derived from the genus Absidia is used.   The method for producing raffinose according to claim 3, wherein the genus Absidia is Absidia corymbifera.   A transformant obtained by transformation with a plasmid containing the DNA of the thermostable α-galactosidase gene represented by the nucleotide sequence of SEQ ID NO: 2 is cultured, and sucrose and galactose are used using the thermostable α-galactosidase obtained from the culture. A method for producing raffinose, comprising producing raffinose using UDP-galactose as a substrate.   The method for producing raffinose according to claim 5, wherein Escherichia coli BL21 (DE3) -pET32Trx / galα (FERM BP-7140) is used as a transformant.

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