JP4060652B2 - Gene encoding cyclodextrin glucanotransferase mainly comprising γ-cyclodextrin and use thereof - Google Patents

Description

【００２４】
本発明の第1のCD組成物の製造方法では、原料溶液の濃度は、例えば、1〜30%の範囲とし、CGTaseをそれぞれ0.05〜300U（乾燥澱粉１ｇ当り）用い、4.5〜12のpH範囲、20〜75℃の温度にて１〜96時間酵素反応を行うことで、α-CD、β-CD及びγ-CDを含有する組成物を生成させることができる。
【００２５】
本発明の第2のCD組成物の製造方法では、澱粉、デキストリン、アミロペクチン及びアミロースからなる群から選ばれる少なくとも１種を含有する原料溶液に本発明の組換えタンパク質であるCGTaseを反応させ、次いでバチルスクラーキ（Bacillus clarkii）種以外の菌株が産生するCGTaseを反応させてα-CD、β-CD及びγ-CDを生成させる。原料溶液の濃度は、例えば、1〜30%の範囲とし、原料溶液に本発明の組換えタンパク質であるCGTaseを0.05〜300U（乾燥澱粉１ｇ当り）加え、4.5〜12のpH範囲、20〜75℃の温度にて１〜96時間酵素反応を行う。次いで、反応液を加熱して本発明の組換えタンパク質であるCGTaseを失活させた後、バチルスクラーキ（Bacillus clarkii）種以外の菌株が産生するCGTase0.05〜300U（乾燥澱粉１ｇ当り）を反応液に加え、4.5〜12のpH範囲、20〜75℃の温度にて１〜96時間酵素反応を行うことで、α-CD、β-CD及びγ-CDを含有する組成物を生成させることができる。
【００２６】
本発明の第３のCD組成物の製造方法では、澱粉、デキストリン、アミロペクチン及びアミロースからなる群から選ばれる少なくとも１種を含有する原料溶液にバチルスクラーキ（Bacillus clarkii）種以外の菌株が産生するCGTaseを反応させ、次いで本発明の組換えタンパク質であるCGTaseを反応させてα-CD、β-CD及びγ-CDを生成させる。原料溶液の濃度は、例えば、1〜30%の範囲とし、原料溶液にバチルスクラーキ（Bacillus clarkii）種以外の菌株が産生するCGTaseを0.05〜300U（乾燥澱粉１ｇ当り）加え、4.5〜12のpH範囲、20〜75℃の温度にて１〜96時間酵素反応を行う。次いで、反応液を加熱してバチルスクラーキ（Bacillus clarkii）種以外の菌株が産生するCGTaseを失活させた後、本発明の組換えタンパク質であるCGTase0.05〜300U（乾燥澱粉１ｇ当り）を反応液に加え、4.5〜12のpH範囲、20〜75℃の温度にて１〜96時間酵素反応を行うことで、α-CD、β-CD及びγ-CDを含有する組成物を生成させることができる。
【００２７】
反応条件(酵素の使用量、反応時間、反応温度等)は、例えば、γ-シクロデキストリンの生成量を１としたときに、α-シクロデキストリンの生成量が0.1〜2の範囲となり、β-シクロデキストリンの生成量が0.1〜2の範囲となるように設定することができる。好ましくは、γ-シクロデキストリンの生成量を１としたときに、α-シクロデキストリンの生成量が0.1〜1の範囲となり、β-シクロデキストリンの生成量が0.1〜0.5の範囲となるように設定する。また、反応条件(酵素の使用量、反応時間、反応温度等)は、例えば、α-シクロデキストリンの生成量を１としたときに、β-シクロデキストリンの生成量が0.1〜1.5の範囲となり、γ-シクロデキストリンの生成量が0.1〜2の範囲となるように設定することもできる。好ましくは、α-シクロデキストリンの生成量を１としたときに、β-シクロデキストリンの生成量が0.3〜0.9の範囲となり、γ-シクロデキストリンの生成量が0.5〜1の範囲となるように設定する。
【００２８】
例えば、本発明の第1のCD組成物の製造方法では、本発明の組換えタンパク質であるCGTaseの使用量に対するバチルスクラーキ（Bacillus clarkii）種以外の菌株が産生するCGTaseの使用量の比を変化させること、CGTaseの最適温度がCGTaseの種類により異なる場合、反応温度を、いずれかのCGTaseの最適温度に近づけることで、各CDの生成比を変化させることで、生成量の比を変化させることができる。本発明の第2のCD組成物の製造方法では、本発明の組換えタンパク質であるCGTaseによるγ-シクロデキストリンを主成分とするCDsの生成条件(酵素の使用量、反応温度、反応時間)と、バチルスクラーキ（Bacillus clarkii）種以外の菌株が産生するCGTaseによるα-シクロデキストリン及び/又はβ-シクロデキストリンを主成分とするCDsの生成条件(酵素の使用量、反応温度、反応時間)とを変化させることで、生成量の比を変化させることができる。
本発明の第3のCD組成物の製造方法では、バチルスクラーキ（Bacillus clarkii）種以外の菌株が産生するCGTaseによるα-シクロデキストリン及び/又はβ-シクロデキストリンを主成分とするCDsの生成条件(酵素の使用量、反応温度、反応時間)と、本発明の組換えタンパク質であるCGTaseによるγ-シクロデキストリンを主成分とするCDsの生成条件(酵素の使用量、反応温度、反応時間)とを変化させることで、生成量の比を変化させることができる。
【００２９】
本発明のCD組成物の製造方法により得られるα-、β-及びγ-シクロデキストリンの混合物を含有する溶液は、さらに精製してシクロデキストリン混合物シラップとすることもできる。この精製は、通常の水飴やオリゴ糖シラップと同様の常法により行うことができる。例えば、この精製は、固形分を除去するための濾過、活性炭処理による脱色、イオン交換樹脂による脱塩を適宜組み合わせて行うことができる。また、本発明の製造方法により得られるα-、β-及びγ-シクロデキストリンを含有する組成物中には、α-，β-およびγ-CDなどの他に、グルコースなどの単糖類、各種オリゴ糖(マルトースなど)或いはデキストリンなども含有している場合があり、これらを常法により適宜分離することもできる。
【００３０】
また、本発明の製造方法により得られるα-、β-及びγ-シクロデキストリンを含有する組成物を含有する溶液を上記のように精製してシクロデキストリン混合物シラップを得たのち、得られたシラップを乾燥してシクロデキストリン混合物粉末を得ることもできる。シラップの乾燥には、スプレードライ法や凍結乾燥法を用いることができる。乾燥法により、結晶状、凍結乾燥状、粉末状、顆粒状などの任意の形態とすることができる。
【００３１】
以下に本発明についてさらに詳細に説明する。
γ -CGTase 遺伝子
上述の様に、本発明によるγ-CGTase遺伝子は、澱粉及びその分解物の中から選ばれた基質に作用し、γ-シクロデキストリン（γ-CD）を主生成物として生成させるシクロデキストリン・グルカノトランスフェラーゼ活性を有するタンパク質をコードするDNA配列であり、γ-CGTaseの部分アミノ酸配列情報をもとに、当該酵素生産菌（バチルスクラーキー7364株）の染色体DNAを鋳型としたPCR法によって当該酵素をコードする遺伝子を取得する事に成功し、さらに大腸菌等の微生物での発現にも成功する事によって決定されたものである。
１）微生物の寄託
本発明の典型的な遺伝子を含むプラスミドpGFT-01（後記載する実施例参照）で形質転換された、本発明γ-CGTaseを著量に発現する大腸菌JM109は、ＧＣＧ３１と命名し、独立行政法人産業技術総合研究所特許生物寄託センターにFERM BP-7648として寄託している。
２）γ -CGTase の酵素学的性質
本発明は、本発明者等によるγ-CGTaseの発見に基づくものであるが、本酵素はバチルスクラーキ7364株から産生されたものであり、その酵素学性質は以下の通りであった（実施例１）。尚、γ-CD生成活性は以下の方法で測定した。
450 μlの1.5 % 可溶性澱粉溶液／25 mM Gly-NaCl-NaOH 緩衝液（pH 10.5）を40℃で保温し、適当に希釈した酵素溶液50 μl を添加することで反応を開始した。反応開始0,5,10,15,20,30 分後にそれぞれ500 μl の0.05 N HClを添加する事で反応を停止した。反応液に5 mM BCG溶液（in 20 % Ethanol） を添加混合後、20 分間室温で保持し、2 ml の１M BCG緩衝液（pH 4.2）を加え630 nm の吸光度を測定した。該吸光度値からあらかじめ作成した検量線を用いて反応液中のγ-CD含量を求めた。γ-CGTase サイクリック活性1 unitを上記反応条件で単位時間当りに1 μmolのγ-CDを生成させる酵素量と定義した。
ａ）BCG法によるγ-CGTase サイクリック活性の測定は、150 mgの可溶性澱粉を10 ml の所定pH 緩衝液（pH3-8: 1/4 × McIlvaine 緩衝液（図１中◆）、 pH8-10.5: 25 mM Gly-NaCl-NaOH緩衝液（図１中■）、 pH 10.5-11.9: 25 mM Na₂HPO₄-NaOH緩衝液（図１中■））に溶解し、その450 μl を基質溶液として用いた。その結果、BCG法によるサイクリック活性の至適pH は10.0-10.5であった（図１）。
【００３２】
ｂ）至適温度
BCG法によるγ-CGTase サイクリック活性の測定は、150 mgの可溶性澱粉を10 ml の 25 mM Gly-NaCl-NaOH緩衝液 （pH 10.0）に溶解し、その450 μl を基質溶液として用いた。所定温度で10 分間反応した。その結果、BCG法によるサイクリック活性の至適温度60 ℃であった（図2）。
ｃ）pH 安定性
BCG法によるγ-CGTase サイクリック活性の測定は、150 mgの可溶性澱粉を10 ml の 25 mM Gly-NaCl-NaOH緩衝液 （pH 10.0）に溶解し、その450 μl を基質溶液として用いた。酵素10 μlに所定のpHの緩衝液90 μlを添加し（a）至適pH 測定に用いた各pH 緩衝液を使用）、4 ℃で24時間放置後、25 mM Gly-NaCl-NaOH緩衝液（pH 10.0）100μl を添加し、その50μlを基質に加え反応を行なった。反応は40 ℃で20分間行なった。
pH 安定性は、pH 6から11まで安定であった（図３）。
【００３３】
ｄ）温度安定性
BCG法によるγ-CGTase サイクリック活性の測定は、150 mgの可溶性澱粉を10 ml の 25 mM Gly-NaCl-NaOH緩衝液 （pH 10.0）に溶解し、その450 μl を基質溶液として用いた。酵素10 μlに25 mM Gly-NaCl-NaOH緩衝液（pH 10.0）を90 μl加え、所定の温度で15分間保持後、その50μlを基質に加え反応を行なった。反応は40 ℃で10分間行なった。本酵素は50℃で15分間処理で、無処理の８０％以上の活性を示した（図4）。
ｅ）分子量
本酵素のSDS-PAGEにより、各種標準タンパク質との相対移動度から求めた分子量は６８KDaであった。
【００３４】
ｆ）等電点
等電点電気泳動により、各種標準タンパク質との相対移動度から求めた等電点は3.98であった。
ｇ）N末端アミノ酸配列
常法により、アプライド・バイオシステムズ製気相プロテインシーケンサー「４７７A型」を使用して分析を行ったところ、本酵素は、N末端に配列番号３に示すアミノ酸配列を有していた。
【００３５】
ｈ）内部アミノ酸配列
常法により、本酵素を断片化し、HPLC法により単離したペプチド断片のN末端配列をアプライド・バイオシステムズ製気相プロテインシーケンサー「４７７A型」を使用して分析を行ったところ配列番号４に示すアミノ酸配列を有していた。
【００３６】
３）γ -CGTase 遺伝子解析
バチルスクラーキ（Bacillus clrkii） 7364の染色体DNAを鋳型として、成熟酵素のN末端アミノ酸配列及び内部アミノ酸配列から設計したプライマーを用いてPCR反応を行った。得られたPCRフラグメントは、γ-CGTaseのN末端から9番目のN以降をコードする1470 bpの遺伝子断片である事が判明した。次いで、染色体DNAを制限酵素Sac Iで完全分解した後、セルフリティゲーション（self-ligation）させて得られた環状DNAを鋳型として、1470 bpのPCRフラグメントの上流配列から設計したアンチセンスプライマー及び下流配列から設計したセンスプライマーを用いてinverse PCR反応を行い、目的酵素遺伝子の全塩基配列を決定した。
【００３７】
４）組換えγ -CGTase 遺伝子の単離・同定
クローニングしたDNA 断片が目的γ-CGTase をコードする遺伝子である事を確認するため、Full F-primer : 5'-gACTTgTACTAAgACAACCTTACg-3' 及びFull R-primer : 5'-gCATCggCTCTACTCATTTCA-3'を用いて、染色体DNAを鋳型としてPCRを行い、γ-CGTase の構造遺伝子、プロモター及び転写終結シグナルを含む2530 bp のPCRフラグメントを得た。次いで、該DNA断片をpGEM-T にligation し、大腸菌JM-109に挿入した。本形質転換大腸菌（pGFT-01/JM109） をLB 培地で16 時間培養後、菌体を破砕し、遠心分離によって不溶物を除去した上澄液はBCG、γ-CD サイクリック 活性で0.35 U/ml の活性を有している事を確認した。更に、本上澄液を粗酵素として、1% 可溶性澱粉（25 mM グリシン-NaCl-NaOH 緩衝液 pH 10）に作用させたところ、HPLCでγ-CDが主生成物であるCGTaseであることを確認した。
次いで、pGFT-01/JM109 をLB 培地で大量培養（600 ml）し、γ-CGTase の精製を行った。精製はγ-CD 結合アフィニティーカラム及びゲル濾過による2段階のプロセスで行い、SDS-PAGE でシングルバンドまで精製した。
本形質転換大腸菌（pGFT-01/JM109）が生産した酵素の諸性質を表２に纏めた。
【００３８】
【表２】

【００３９】
本形質転換大腸菌が生産したγ-CGTaseのN末端配列を常法により、アプライド・バイオシステムズ製気相プロテインシーケンサー「４７７A型」を使用して分析を行ったところ、本酵素は、N末端に配列番号３に示すアミノ酸配列を有していた。
この組換え型γ-CGTaseの酵素学的特性はバチルスクラーキー7364由来のγ-CGTaseとほぼ一致した。従って、前述の組換えγ-CGTaseをコードするDNAはバチルスクラーキー7364由来のものであると判断した。
【００４０】
５）組換えγ -CGTase をコードする遺伝子の発現／製造
ａ）発現ベクター
本発明によるγ-CGTaseをコードする断片を、宿主細胞内で複製可能であるか、あるいは染色体に組み込まれかつ同遺伝子が発現可能な状態で含むDNA分子、特に発現ベクターの形態として宿主細胞の形質転換を行えば、宿主細胞において本発明によるγ-CGTaseを産生する事ができる。従って、本発明によれば、本発明によるγ-CGTaseをコードする遺伝子を含んだDNA分子、特に発現ベクターが提供される。好ましくはこのベクターはプラスミドである。
【００４１】
本発明において利用されるベクターは、使用する宿主細胞によってウイルス、プラスミド、コスミドベクター等から適宜選択可能である。例えば、宿主細胞が枯草菌の場合はｐUB系のプラスミド、大腸菌の場合はλファージ系のバクテリオファージ、ｐBR322、BluesscriptIISK(+), pUC18, pUC19, pUC118, pUC119, pGEM-T pCR2.1, pLEX, pJL3, pSW1, pSE280, pSE420, pHY300PLK 等のプラスミドベクターが上げられるが、大腸菌で発現するには、ｐBR322、BluesscriptIISK(+), pGEM-T、pUC18, pUC19, pUC118, pUC119, pCR2.1が好適であり、枯草菌で発現するにはpHY300PLKが好適である。ｐBR、ｐUC系プラスミド、酵母の場合はYep,Ycp系、YIP系ベクターなどが挙げられる。このプラスミドは形質転換体の薬剤耐性や栄養要求マーカーなどの選択マーカーを含むものが好ましい。さらに、発現ベクターとしては、γ-CGTase遺伝子の発現に必要なプロモーター、ターミネーター、リボソーム結合部位、転写終結シグナルなどのDNA配列を有している事が好ましい。
【００４２】
プロモーターとしては、大腸菌においては一般に慣用されるlac、 trp、 tac、T7等のプロモーターが好ましく用いる事ができるし、野生株のγ-CGTaseの発現に使用されているプロモーターをそのまま用いても発現が可能である。配列番号２に示しているアミノ酸配列の１番から702番までの配列にはシグナルペプチドを含んでいるが、後記の実施例に示すようにこの配列をそのまま用いても構わない。又、枯草菌においては、キシロースオペロン、ズブチリシン、SPACなどのプロモーター、酵母ではADH、PHO、GAL、GAP等のプロモーターが好ましく用いる事ができる。
【００４３】
ｂ）形質転換体／培養
宿主としては、大腸菌、枯草菌、放線菌、酵母などが挙げられ、形質転換体の培養条件でアミラーゼを産出しない物であれば良い。本発明によるCGTaseの製造方法は、上記形質転換細胞を培養し、この培養物中から、澱粉及びその分解物の中から選ばれた基質に作用し、γ-シクロデキストリン（γ-CD）を主生成物として生成させるシクロデキストリン・グルカノトランスフェラーゼ活性を有するタンパク質を採取する事を特徴とするものである。又、本発明による組換えγ-CGTaseは上記遺伝子の発現産物である。即ち、本発明によるγ-CGTaseは、配列番号２のアミノ酸番号１から７０２で示されるアミノ酸配列又は配列番号２のアミノ酸番号２９から７０２で示されるアミノ酸配列と実質的に同一のアミノ酸配列（該アミノ酸配列またはその１もしくは複数のアミノ酸配列が置換、欠失、挿入もしくは付加されたアミノ酸配列）を有し、かつ例えば澱粉デキストリン、アミロペクチン又はアミロースに作用し、γ-シクロデキストリン（γ-CD）を主生成物として生成させる（即ち、主としてγ-シクロデキストリンを生成し、β-及びα-シクロデキストリンの生成量はγ-シクロデキストリンの生成量と比較して低い）シクロデキストリン・グルカノトランスフェラーゼ活性を有するタンパク質である。
【００４４】
本発明の形質転換体の培養に用いる栄養培地としては、炭素源、窒素源、無機物及び必要に応じ使用菌株の必要とする微量栄養素を程よく含有する物であれば、天然培地、合成培地のいずれでもよい。炭素源としては、グルコース、マルトースなどのマルトオリゴ糖、フラクトース、澱粉、デキストリン、グリセリンなどの炭化水素が挙げられる。窒素源としては、塩化アンモニウム、硫酸アンモニウム、尿素、硝酸アンモニウム、硝酸ナトリウム、グルタミン酸などのアミノ酸、尿素などの無機窒素有機窒素化合物が用いられる。更に、ペプトン、ポリペプトン、肉エキス、酵母エキス、CSL、大豆粉、大豆粕、乾燥酵母、カザミノ酸などの窒素含有有機天然物も使用可能である。無機物としては、リン酸二水素カリウム、リン酸水素二カリウム、硫酸マグネシウム、硫酸第一鉄、硫酸マンガン、硫酸亜鉛、塩化ナトリウム、塩化カリウム、塩化カルシウム等が用いられる。その他にビオチン、チアミン等の微量栄養素を必要に応じて使用する。培養法としては液体培養法がよく、工業的には通気攪拌培養法が適している。培養温度とpHは使用する形質転換体の増殖にもっとも適した条件を選べば良い。培養時間は培養時間によって変わってくるが、組換えγ-CGTaseの生成が確認された時、好ましくは生成量が最大に達した時に培養を停止する。この様にして得られた培養物から本発明の組換えγ-CGTaseを採取するには、先ず、培養液中の菌体を超音波などの物理的手法で破砕するか、有機溶剤やリゾチーム等の酵素によって細胞膜を溶菌した後、残査を遠心分離や濾過法によって除去する。これを限外膜濾過、塩析、溶剤沈澱法などの処理を行う事によって工業用用途の濃縮粗酵素液が調製できる。さらに、本濃縮酵素をγ-CDを固定化させたアフィニティークロマトグラフィー、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、等の周知の単離精製方法を組み合わせる事により、容易に精製酵素標品を得る事ができる。
【００４５】
ｃ）γ-シクロデキストリン（γ-CD）及び所望のCDバランス（α-、β-及びγ -CDバランス）を有するCD含有組成物の製造方法
本発明によれば、上記のγ-CGTase を用いたγ-シクロデキストリン（γ-CD）及び所望のCDバランス（α-、β-及びγ-CDバランス）を有するCD含有組成物の製造方法が提供される。即ち、本発明方法によりCDを製造する為には、例えば、1〜30%の澱粉（澱粉又はその組成画分、加工澱粉などを含む）を含有する水溶液に本組換えγ-CGTase酵素液（精製酵素又は粗酵素）単独もしくは他起源の微生物が産生するCGTase（α-及びβ-CGTase）を0.1〜300U（乾燥澱粉１ｇ当り）併用添加し、pH4.5から12、温度20から60℃にて１〜96時間酵素反応を行う。尚、澱粉は必要に応じ、予め加熱し、液化処理を施して用いる。
【００４６】
上述のような方法で調製した糖類 (含有組成物)中には、α-，β-およびγ-CDなどの他に、グルコースなどの単糖類、各種オリゴ糖(マルトースなど)或いはデキストリンなども含有している場合がある。また、必要に応じて所望の単一重合度を有するCDを分離(結晶化、クロマト分画、酵母などによる醗酵処理及び酵素処理などによる)して用いることもできる。なお形態は、上述の方法で得られるような含有組成物状の他、結晶状、凍結乾燥状、粉末状、顆粒状などの任意の形態であることはいうまでもない。本発明によるCDは、これまで市販されているCDと同様に経口摂取可能な全ての飲食品、例えば、茶類、清涼飲料などの飲料類、キャンディー、ゼリー、和菓子などの和洋菓子類、ヨーグルト、アイスクリームなどの乳製品類、ハム、ソーゼージなどの畜肉加工品類、蒲鉾、なると等の水産加工品類や麺類、漬物類、その他調理済加工食品類やインスタント食品類などに、香料などの安定化や乳化作用および賦形剤などとして添加・共存させ、安全な食品添加物であるCDを使用することにより、極めて簡便に且つ効果的に飲食品の嗜好性及び機能性を向上させるものである。また、飲食品のみならず医薬品や化粧料などの有効成分の安定化や乳化作用、賦形剤などとしても利用することが可能である。
【００４７】
本発明のCDの使用方法は、飲食品、医薬品や化粧料中にCDが共存する条件であれば特に限定されるものではない。例えば、CDを基礎となる飲食品素材加工中に同時に添加しても、或いは基礎飲食品加工終了後に添加してもよく、各種食品の製造工程の実状に適した添加方法を用いればよい。CDの添加量は、飲食品の場合、基礎となる飲食品本来の味質並びに風味を損なわない限り特に限定はないが、一般に20重量％以下の添加量で添加するのが好ましい。20重量％以上では、CDが有するマスキング効果により、基礎となる飲食品の味質或いは風味を変えて、嗜好性を減じてしまう懸念があるので好ましくはない。医薬品や化粧料の場合には、有効成分の効果効能を損なわない限り特に限定はない。
【００４８】
【実施例】
以下に本発明の実施例を示すが、これは本発明を更に具体的に説明する為のものであり、本発明が以下の実施例の範囲のみに限定されるものではない。
【００４９】
本酵素はバチルスクラーキ7364株から産生されたものであり、以下の方法で調製した。
バチルスクラーキ7364株(FERM BP-7156)を炭素源としてネオタック #30T（日本食品化工（株）製） 1.0 % (w/v)、窒素源としてソヤフラワーFT（日清製油製）0.5 %及び酵母エキス（Difco 製）0.5 %、K₂HPO₄ 0.1 %、MgSO₄・7H₂O 0.02 %及びNa₂CO₃ 0.8 % を含む液体培地で37 ℃、48 時間振とう培養するとその培養液中にCGTaseを分泌した（Blue value 法 20 U/ml 培養上澄）。
得られたCGTaseをアフィニティークロマトグラフィー法により精製した。
【００５０】
γ-CGTase活性は以下の方法でγ-CD生成活性（環状化活性）として測定した。即ち、450 μlの1.5 % 可溶性澱粉溶液／25 mM Gly-NaCl-NaOH 緩衝液（pH 10.5）を40℃で保温し、適当に希釈した酵素溶液50 μl を添加することで反応を開始した。反応開始0,5,10,15,20,30 分後にそれぞれ500 μl の0.05 N HClを添加する事で反応を停止した。反応液に5 mM BCG溶液（in 20 % Ethanol） を添加混合後、20 分間室温で保持し、2 ml の１M BCG緩衝液（pH 4.2）を加え630 nm の吸光度を測定した。該吸光度値からあらかじめ作成した検量線を用いて反応液中のγ-CD含量を求めた。γ-CGTase サイクリック活性1 unitを上記反応条件で単位時間当りに1 μmolのγ-CDを生成させる酵素量と定義した。
【００５１】
（実施例１） 精製γ-CGTaseの酵素化学的特性
実施例1-1 精製酵素の調製
バチルスクラーキ7364株の培養及び該菌株の産生するγ-CGTaseの精製はアフィニティークロマトグラフィー法により行った。
【００５２】
実施例1-2 精製酵素の酵素学的特性
１）作用
可溶性澱粉を基質として、10 %(w/v) になるように50 mM グリシン-NaCl-NaOH (pH 10.0) 緩衝液に溶解したものを基質溶液とした。基質溶液5 ml に上記精製酵素を0.5 U/g DSになるように添加し、50 ℃で48時間反応させた。反応生成物を特願2000-151056に記載したHPLC法で調べたところ、48 時間後ではγ-CD が9.7 %、α- 及びβ-CD はそれぞれ1.7及び0.9 % （HPLC area）生成した。
【００５３】
２）至適pH
BCG法によるγ-CGTase サイクリック活性の測定は、150 mgの可溶性澱粉を10 ml の所定pH 緩衝液（pH3-8: 1/4 × McIlvaine 緩衝液 pH8-10.5: 25 mM Gly-NaCl-NaOH緩衝液 pH 10.5-11.9: 25 mM Na₂HPO₄-NaOH緩衝液）に溶解し、その450μl を基質溶液として用いた。その結果、BCG法によるサイクリック活性の至適pH は10.0-10.5であった（図１）。
【００５４】
３）至適温度
BCG法によるγ-CGTase サイクリック活性の測定は、150 mgの可溶性澱粉を10 ml の 25 mM Gly-NaCl-NaOH緩衝液 （pH 10.0）に溶解し、その450 μl を基質溶液として用いた。所定温度で10 分間反応した。その結果、BCG法によるサイクリック活性の至適温度60 ℃であった（図2）。
【００５５】
４）pH 安定性
BCG法によるγ-CGTase サイクリック活性の測定は、150 mgの可溶性澱粉を10 ml の 25 mM Gly-NaCl-NaOH緩衝液 （pH 10.0）に溶解し、その450 μl を基質溶液として用いた。酵素10 μlに所定のpHの緩衝液90 μlを添加し（a）至適pH 測定に用いた各pH 緩衝液を使用）、4 ℃で24時間放置後、25 mM Gly-NaCl-NaOH緩衝液（pH 10.0）100μl を添加し、その50μlを基質に加え反応を行なった。反応は40 ℃で20分間行なった。
pH 安定性は、pH 6から11まで安定であった（図３）。
【００５６】
５）温度安定性
BCG法によるγ-CGTase サイクリック活性の測定は、150 mgの可溶性澱粉を10 ml の 25 mM Gly-NaCl-NaOH緩衝液 （pH 10.0）に溶解し、その450 μl を基質溶液として用いた。酵素10 μlに25 mM Gly-NaCl-NaOH緩衝液（pH 10.0）を90 μl加え、所定の温度で15分間保持後、その50μlを基質に加え反応を行なった。反応は40 ℃で10分間行なった。本酵素は50℃で15分間処理で、無処理の８０％以上の活性を示した（図4）。
【００５７】
６）分子量
本酵素のSDS-PAGEにより、各種標準タンパク質との相対移動度から求めた分子量は６８KDaであった。
【００５８】
７）等電点
等電点電気泳動により、各種標準タンパク質との相対移動度から求めた等電点は3.98であった。
【００５９】
８）N末端アミノ酸配列
常法により、アプライド・バイオシステムズ製気相プロテインシーケンサー「４７７A型」を使用して分析を行ったところ、本酵素は、N末端に配列番号３に示すアミノ酸配列を有していた。
【００６０】
９）内部アミノ酸配列
常法により、本酵素を断片化し、HPLC法により単離したペプチド断片のN末端配列をアプライド・バイオシステムズ製気相プロテインシーケンサー「４７７A型」を使用して分析を行ったところ配列番号４に示すアミノ酸配列を有していた。
【００６１】
（実施例２）
実施例２−１ 染色体DNAの調製
バチルスクラーキ（Bacillus clarkii） 7364 の染色体DNAはN. Declerck等の報告（N. Declerck, P. Joyet, D. Le Coq and H. Heslot, J. Biotech., 8, 1998, 23-38）を参考に以下のように調製した。バチルスクラーキ（Bacillus clarkii） 7364株を酵素生産培地にて培養した培養液（40 ml）より遠心分離によって（8,000 rpm, 10 min）菌体を集めた。得られた菌体を5 ml のTESS緩衝液（30 mM Tris/HCl (pH 7.5), 5 mM EDTA, 50 mM NaCl, 25 % sucrose）で2 回洗浄後、3 mlの同緩衝液に懸濁した。菌体懸濁液を65 ℃で10 分間処理し、37 ℃まで冷却した。リゾチーム水溶液（50 mg/ml）を1 ml 添加し、37 ℃で1時間反応した。次いで、プロテイナーゼ K を25 mg/ml になる水に溶解後、1 時間37 ℃で自己消化させその500 μl 添加し、同温度で2時間反応した。10 % SDS 1 mlを加え、65 ℃で10 分間処理する事で反応を停止した。反応液をTE 飽和フェノール及びフェノール/クロロフォルム（2 回）で処理し、エタノール沈澱によって回収した染色体DNAを1.2 ml のTEに溶解後、RNAase溶液（500 μg/ml）を10 μg/mlになるように添加し、37 ℃で2時間反応した。フェノール/クロロフォルム（2 回）で処理し、エタノール沈澱によって染色体DNAを回収した。本操作によって162 μgの染色体DNAを得た。
【００６２】
実施例２−２ γ-CGTase をコードするDNA断片の取得と塩基配列の決定
Plasmidsの調製、制限消化、ライゲーション及び大腸菌の形質転換は既報の方法に準じて行った（Sambrook, J., Fritsch, E. F. & Maniatis, T. (1982) Molecular cloning: a laboratory manual, 2^nd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York）。塩基配列の決定はベックマン・コールター（Beckman Coulter ）社のマルチキャピラリーDNA解析システムCEQ2000を用いたダイデオキシ・チェイン・ターミネーション（the dideoxy chain termination）法で行った。DNA鎖の塩基配列の決定及びコンピューター解析はGENETYX-WINを用いて行った。
γ-CGTaseをコードする遺伝子の部分断片は、染色体DNAを鋳型として、N-terminal F primer：5'-AAYgTIAAITAYgCIgARgARgT-3'及びDomain Cr primer：5'-gCRTCICCIggYTTICCCATDCC-3' を用いてPCRを行う事で調製した。これらプライマーは、N-terminal F primerについてはエドマン法で決定した成熟蛋白のN末端アミノ酸配列の内、9番目以降のN-V-N-Y-A-E-E、Domain Cr primer については成熟蛋白のプロテアーゼによるランダム分解より得られたペプチド断片のN末端アミノ酸配列の一部PMGKPGDAに基づき設計した。以下にPCRの反応条件を纏めた。
【００６３】
【表３】

【００６４】
上記反応液について、94 ℃で5 分間加熱した後、94 ℃で1分間、51 ℃で2 分間、72 ℃で3分間のサイクルを30回繰り返してから、最後に72 ℃で10分間保温した。本PCRによって得られた約1500 bp のフラグメントをT-ベクター pGEM-Tにライゲーション後、塩基配列を決定した。該塩基配列から推定されるアミノ酸配列中には、エドマン分析から得られたN末端アミノ酸配列（成熟酵素N末端アミノ酸配列及び酵素内部ペプチドN末端配列）と完全に一致する配列が認められ、更にアミラーゼファミリー酵素群のコンセンサス配列を見出す事ができた。そこで、該遺伝子断片はγ-CGTase成熟酵素蛋白のN末端から9番目以降をコードする1470 bpの断片であると断定した。
【００６５】
上記実施例で調製した染色体DNAを制限酵素Sac I, EcoRI、BamHI 等で完全消化し、アガロースゲル電気泳動で分離した。分離したDNA断片を常法によりナイロン膜に転写し、上記１４７０bpのフラグメントを得る際に行ったPCRにおいて、ｄNTPと１：１の割合でDig標識したｄNTPを混同し、同様の条件でPCRを行う事で調製したDig標識1470bp DNA断片を用いて、前述ナイロン膜に固定したDNA断片とサザンハイブリダイズさせた（Southern et al, J. Mol. Biol., 98, 503-517, 1975）。 条件は５× SSC、１％SDS、１０％デキストラン、０．５mg /ml サケ変性DNAからなる溶液中、６５℃で１６−２０時間ハイブリダイズを行い、その後、２× SSC、１％SDSからなる溶液中、６５℃で30分間3回洗浄した。その結果、SacIで消化したDNA断片の約２５００ｂｐの断片がハイブリダイズした。
【００６６】
次いで、該フラグメントの5'-及び3'-上流及び下流領域を取得するため以下の実験を行った。先ず、染色体DNAをSac Iで完全消化した後、消化物をモノメリックサークル（monomeric circles）が生成する様適当な条件下でT4DNA リガーゼを加え、16 ℃で一昼夜反応させた（消化染色体DNA 1μg、10 ×ライゲーション・バッファー 10 μl、T4 リガーゼ700 U／1 ml ）。得られたSac I-self-circulized DNA 分子を鋳型として、inverse anti primer：5'-gATCTgTTACAATATgATAAAT-3'及びinverse sense primer：5'-TTATTAGACggTCAATCGTTA-3' を用いてPCRを行った。これらプライマは、inverse anti primerについては前述1470 bpのフラグメントの5'-上流域から、inverse sense primerについては前述1470 bpフラグメント3'-下流の塩基配列に基づき設計した。以下にPCRの反応条件を纏めた。
【００６７】
【表４】

【００６８】
上記反応液について、94 ℃で5 分間加熱した後、94 ℃で0.5分間、47 ℃で0.5 分間、72 ℃で0.5分間のサイクルを10回、94 ℃で0.5分間、50 ℃で0.5 分間、72 ℃で1分間のサイクルを10回、94 ℃で1分間、50 ℃で2 分間、72 ℃で3分間のサイクルを10回繰り返してから、最後に72 ℃で10分間保温した。本PCRによって得られた約2000 bp のフラグメントをT-ベクター pGEM-Tにligation後、塩基配列を決定した。その結果、γ-CGTase の残りのN末端配列及び開始コドン、リボソーム結合領域（RBS）及びプロモーター配列を含む約0.4 Kbp の上流域塩基配列及び残りのC末端領域及び終始コト゛ン、inverted repeatを含む約0.2 Kbp の下流域の塩基配列を決定し、配列番号１に示す塩基配列が決定された。
【００６９】
実施例2-3 組換えプラスミドpGFT-01 及び大腸菌の形質転換
配列番号１ に示したγ-CGTase 遺伝子及びそのフランキング領域（Flanking region） を染色体DNAを鋳型として、Full F-primer : 5'-gACTTgTACTAAgACAACCTTACg-3' 及びFull R-primer : 5'-gCATCggCTCTACTCATTTCA-3'をprimerとしてPCRを行った。反応に用いたprimerは開始コドンから237 bp 上流配列及び終始コドンから77 bp下流の塩基配列から設計した。
以下にPCRの反応条件を纏めた。
【００７０】
【表５】

【００７１】
上記反応液について、94 ℃で5 分間加熱した後、94 ℃で1.0分間、55 ℃で1.5 分間、72 ℃で3分間のサイクルを32回繰り返してから、最後に72 ℃で10分間保温した。
得られた約2.5 KbpのDNA断片をT-ベクター pGEM-Tにライゲーション及び大腸菌JM109へ挿入後、プラスミドｐGFT-01）を大量調製し、プライマーウォーキング（primer walking） 法によって全塩基配列の確認を行い、少なくとも配列番号１に示す塩基配列を有するDNAが含まれていることを確認した。
【００７２】
実施例３ 大腸菌が生産するγ-CGTaseの精製と酵素学的性質
pGFT-01/JM109 をLB 培地で大量培養（600 ml）し、γ-CGTase の精製を行った。精製は上記実施例１の方法に準じ、γ-CD 結合アフィニティーカラム及びゲル濾過による2段階のプロセスで精製が可能であり、SDS-PAGE でシングルバンドまで精製した。15.5mg(79.8U)のタンパク質が得られた(回収率73%)。
本形質転換大腸菌（pGFT-01/JM109）が生産した酵素の諸性質を表６に纏めた。
【００７３】
【表６】

バチルスクラーキ（B. clarkii）の生産する酵素(1)及び大腸菌生産酵素(2)のSDS-PAGEの結果を図5に示す。大腸菌生産酵素の諸性質は、バチルスクラーキ（B. clarkii）の生産する酵素とほぼ同様な諸性質を示した。
【００７４】
（実施例４）大腸菌が生産するγ-CGTaseを用いたCD含有組成物の調製（１）
コーンスターチを常法によりα−アミラーゼを用いて液化させ、濃度20重量％、グルコース当量７の澱粉液化液を調製した。次いでこの澱粉液化液をpH７に調整した後、実施例３記載の大腸菌産生γ-CGTaseをUF 濃縮膜（PM-10）を用いて濃縮したものを粗酵素液として、１単位/g基質添加し、55℃，48時間反応させた。その後、本反応液を加熱し酵素を失活させ、脱色、イオン交換などの精製を行いCDを含む含有組成物を調製した。その糖組成をHPLC 法で求めた。
【００７５】
【表７】

【００７６】
（実施例 ５）大腸菌が生産するγ-CGTaseを用いたCD含有組成物の調製（２）
コーンスターチを常法によりα-アミラーゼを用いて液化させ、濃度10重量％、グルコース当量７の澱粉液化液を調製した。次いでこの澱粉液化液をpH 7に調整した後、実施例３記載の大腸菌産生γ-CGTaseをUF 濃縮膜（PM-10）を用いて濃縮したものを粗酵素液として2単位／ｇ基質添加し、55℃，48時間反応させた。その後、本反応液を90℃まで加熱し酵素を失活させ、75℃まで冷却後Bacillus属由来のCGTase(日本食品化工(株)製)を40単位／ｇ基質添加し、24時間反応させた。90℃まで加熱し酵素を失活させ、脱色、イオン交換などの精製を行いCDを含む含有組成物を調製した。その糖組成をHPLC 法で求めた。
【００７７】
【表８】

【００７８】
（実施例 ６）
コーンスターチを常法によりα-アミラーゼを用いて液化させ、濃度10重量％、グルコース当量７の澱粉液化液を調製した。次いでこの澱粉液化液をpH 7に調整した後、実施例３記載の大腸菌産生γ-CGTaseをUF 濃縮膜（PM-10）を用いて濃縮したものを粗酵素液として３単位／ｇ基質、および20単位／ｇ基質のBacillus属由来のCGTase(日本食品化工(株)製)を同時に添加し、55℃，72時間反応させた。その後、本反応液を90℃まで加熱し酵素を失活させ、脱色、イオン交換などの精製を行いCDを含む含有組成物を調製した。その糖組成をHPLC 法で求めた。
【００７９】
【表９】

【００８０】
【発明の効果】
本発明は、本発明者等が見出した好アルカリ性バチルス属細菌、特にバチルスクラーキー７３６４由来のγ-CGTase遺伝子を基に、遺伝子工学的手法により、組換えγ-CGTase生産微生物を作製したものであって、これを培養すれば酵素の生産性の改善と不純物の少ないγ-CGTaseを産生させる事が可能になった点で、極めて優れている。又、本組換え酵素を工業的規模で生産することが可能になった結果、γ-シクロデキストリン（γ-CD）及び所望のCDバランス（α-、β-及びγ-CDバランス）を有するCD含有組成物の製造が飛躍的に効率よく製造する事ができる点においても、非常に価値がある。
【配列表】

【図面の簡単な説明】
【図１】BCG法によるγ-CGTase サイクリック活性に対するpH の影響。
【図２】BCG法によるγ-CGTase サイクリック活性に対する温度の影響。
【図３】BCG法によるγ-CGTase サイクリック活性に対するpH安定性。
【図４】BCG法によるγ-CGTase サイクリック活性に対する温度安定性。
【図５】バチルスクラーキ（B. clarkii）の生産する酵素及び大腸菌生産酵素のSDS-PAGEの結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cyclodextrin / glucanotransferase that acts on a starch and a substrate selected from starch degradation products such as dextrin, amylopectin and amylose to produce γ-cyclodextrin (γ-CD) as a main product. Γ-cyclodextrin (γ-CD), which acts on a substrate selected from a recombinant plasmid containing the gene, a transformant thereof, a transformant thereof, and starch and a degradation product thereof using the transformant. ) As a main product, a method for producing cyclodextrin / glucanotransferase, and γ-cyclodextrin (γ-CD) and a desired CD balance (α-, β using the recombinant cyclodextrin / glucanotransferase) -And γ-CD balance).
[0002]
[Prior art]
Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) acts on α-1,4-glucan such as starch, and cyclodextrin (CD) is a cyclic α-1,4-glucan due to its intramolecular transfer activity Is an enzyme that produces The degree of polymerization of CD produced by CGTase is mainly 6-8, and is called α-, β- and γ-CD, respectively. In addition to this CD formation reaction, CGTase is coupled through an intermolecular transfer reaction, a coupling reaction (transferring a linear oligosaccharide resulting from ring opening of CD to a receptor sugar molecule) and a disproportionation reaction. Catalyze (transfer of linear oligosaccharide to acceptor sugar molecule). Furthermore, although CGTase is weak, it also catalyzes the hydrolysis reaction of α-1,4-glucoside bond. CGTase is positioned as an important enzyme in the food, pharmaceutical and cosmetic industries because CD can make inclusions with many molecules and change its chemical and physical properties. Therefore, it started in 1939 with CD synthesis reaction by Bacillus macerans enzyme (EB Tilden and SJ Pirt, J. Am. Chem. Soc., 63, 2900-2902, 1939), and then CGTase producing bacteria Numerous studies have been conducted, including search for proteins and enzyme purification (Sumio Kitahata, Naoto Tsuyama and Shigetaka Okada, Agr. Biol. Chem., 38 (2), 387-393, 1974, Sumio Kitahata and Shigetaka Okada , Agr. Biol. Chem., 38 (12), 2413-2417, 1974, Sumio Kitahata and Shigetaka Okada, J. Jap. Soc. Starch Sci., 29 (1), 13-18, 1982, Michio Kubota, Yoshiki Matsuura, Shuzo Sakai and Yukiteru Katsube, Denpun Kagaku, 38 (2), 141-146, 1991, Lionel J. Bovetto, Daniel P. Backer, Jaques R. Villette, Philippe J. Sicard, and Stephane JL. Bouquelet, Biotechnology and Applied Biochemstry, 15, 48-58, 1992, Shinske Fujiwara, Hirofumi Kakihara, Kim Myung Woo, Andre Lejeune, Mitsuhide Kanemoto, Keiji Sakaguchi, and Tadayuki Imanaka, Applied and environmental microbiology, 58 (12), 4016-4025, 1992, Florian Binder, Otto Huber and August Bock, Gene, 47, 269-277, 1986, Keiji Kainuma, Toshiya Takano and Kunio Yamane, Appl. Microbiol. Biotechnol., 26, 149-153, 1987, Takahiro Kaneko, Tetsuo Hamamoto and Koki Horikoshi, J. general Microbiology, 134, 97-105, 1988, Murai Makela, Pekka Mattsson, M. Eugenia Schinina, and Timo Korpela, Biotechnology and Applied biochemistry, 10, 414-427, 1988, Ernest KC Yu, Hiroyuki Aoki, and Masanaru Misawa, Appl. Microbiol. Biotechnol., 28, 377-379, 1988).
[0003]
This CGTase is classified as α-, β-CGTase and γ-CGTase depending on the type of main CD synthesized. Many of the previously reported genes are α- or β-CGTase, and few enzymes have been reported as γ-CGTase (Shigeharu Mori, Susumu Hirose, Takaichi Oya, and Sumio Kitahata, Oyo Toshitsu Kagaku, 41 (2), 245-253, 1994, Yoshito Fujita, Hitoshi Tsubouchi, Yukio Inagi, Keiji Tomita, Akira Ozaki, and Kazuhiro Nakanishi, J. Fermentation and Bioengineering, 70 (3), 150-154, 1990, Takashi Kato and Koki Horikoshi, J. Jpn. Soc. Starch Sci., 33 (2), 137-143, 1986). In addition, even in the enzyme reported as γ-CGTase, the amount of γ-CD produced is 5% or less, or the rate of β-CD production is accelerated in the later stage of the reaction, and the same amount or more than γ-CD. β-CD was produced, or the production of γ-CD was significantly reduced at substrate concentrations of 10% or more, and this was not an industrially available enzyme, for example, by coexisting ethanol in the reaction solution. .
[0004]
On the other hand, attempts have been made to improve the production of γ-CD by modifying the structural gene of α- or β-CGTase (Akira Nakamura, Keiko Haga, and Kunio Yamane, Biochemstry, 32, 6624-6631, 1993). Michio Kubota, Yoshiki Matsuura, Shuzo Sakai and Yukiteru Kutsume, Oyo Toshitsu Kagaku, 41 (2), 245-253, 1994). However, even when the amount of γ-CD produced is increased, β-CD produced by the original activity is not significantly reduced, and it cannot be said that it is sufficient from an industrial viewpoint. Therefore, α-CD and β-CD are used in various fields, but γ-CD is rarely performed. The same applies to a CD-containing composition, and a CD-containing composition containing α- or β-CD as a main component is used in various fields, but a CD-containing composition containing γ-CD as a main component is used. Few. In a CD-containing composition, the CGTase used to prepare the CD composition is determined by α-, β-, or γ-CGTase, making it difficult to prepare a CD-containing composition having a desired CD balance. .
[0005]
In view of such a situation, the present inventors have found that the Bacillus clarkey 7364 produces a novel γ-CGTase having γ-CD as the main product. A method for producing a CD-balanced composition containing CD balance has been completed, and patent applications have been filed previously (Japanese Patent Laid-Open Nos. 2001-327299 and 2001-327284).
[0006]
However, these microorganisms cannot be said to have sufficient enzyme productivity, and when preparing a CD-containing composition of γ-CD and a desired CD balance on an industrial scale, there is a problem that the microorganisms must be cultured in large quantities. there were. To solve this problem, in order to improve the enzyme productivity of microorganisms, wild-type strains have been treated with ultraviolet rays, X-rays, NTG (N-methyl-N'-nitro-N-nitrosoguanidine) and EMS (ethyl methanesulfonate). Such a method has been used that a complicated breeding operation using a chemical such as the above is carried out to produce a mutant strain having improved enzyme productivity. Furthermore, microorganisms producing CGTase often produce a small amount of α-amylase simultaneously with CGTase. For this reason, when CGTase is allowed to act on a substrate selected from starch and its degradation products, and γ-cyclodextrin (γ-CD) is produced as the main product, γ-amylase mixed in the crude enzyme produces γ -Yield decreases due to hydrolysis of CD. One way to solve this problem is to purify the crude enzyme to remove amylase. In this case, however, there is a problem that the enzyme production cost increases. As another method, a method of relatively increasing the CGTase activity by suppressing the expression of the amylase gene by an artificial technique can be considered. However, even in this method, it is often difficult to obtain a mutant strain that selectively suppresses only amylase activity.
[0007]
On the other hand, it is now possible to easily obtain a gene encoding a useful enzyme, and a desired amount of enzyme can be obtained relatively easily by preparing a recombinant DNA containing this gene and introducing it into a microorganism. I can do it now.
[0008]
In view of such circumstances, it is an important technique to find the gene encoding the γ-CGTase, analyze the gene sequence, and improve the productivity and activity of the enzyme by a transformant incorporating the gene. Furthermore, if a gene can be obtained, a highly active enzyme can be obtained by preparing a mutant, and further, heat resistance, pH resistance, and reaction rate have been increased using protein engineering techniques. You can also expect to get enzymes.
[0009]
[Problems to be solved by the invention]
Therefore, the present invention relates to a gene encoding cyclodextrin / glucanotransferase that acts on a substrate selected from starch and its degradation product and generates γ-cyclodextrin (γ-CD) as a main product, the gene Recombinant plasmid containing sucrose and its transformant, and acts on a substrate selected from starch and its degradation product using the transformant to produce γ-cyclodextrin (γ-CD) as the main product Of cyclodextrin / glucanotransferase to be produced, and γ-cyclodextrin (γ-CD) and desired CD balance (α-, β- and γ-CD balance) using the recombinant cyclodextrin / glucanotransferase It aims at providing the manufacturing method of the CD containing composition which has this.
[0010]
[Means for Solving the Problems]
As a result of searching for a novel γ-CGTase having γ-CD as a main product from nature, the present inventors have found that the desired γ-CGTase is produced by an alkalophilic Bacillus bacterium (Japanese Patent Laid-Open No. 2001-327299). No. 1, JP 2001-327284 A), the alkaliphilic Bacillus bacterium isolated from the rot of starch is named Bacilscleraki 7364 strain, and FERM BP- Deposited as 7156.
As a result of further earnest research, we succeeded in isolating the γ-CGTase gene of this strain and succeeded in expression in microorganisms such as Escherichia coli. The present invention has been completed based on these findings.
[0011]
The present invention is as follows.
(Claim 1) A DNA encoding a protein having an amino acid sequence represented by amino acid numbers 1 to 702 of SEQ ID NO: 2 shown in the sequence listing.
(Claim 2) DNA encoding a protein having an amino acid sequence represented by amino acid numbers 29 to 702 of SEQ ID NO: 2 shown in the sequence listing.
(Claims3) DNA having the base sequence of 321 to 2426 bases or the base sequence of 405 to 2426 bases of SEQ ID NO: 1 shown in the sequence listing.
(Claims4Claim 1~ 3Hybridizes with a DNA comprising a base sequence complementary to the DNA of any one of 1) under stringent conditions, and acts on starch, dextrin, amylopectin or amylose to produce mainly γ-cyclodextrin, A DNA encoding a protein having cyclodextrin / glucanotransferase activity in which the amount of β- and α-cyclodextrin produced is lower than that of γ-cyclodextrin.
(Claims51) The DNA is derived from a bacterium belonging to the genus Bacillus.4DNA of any one of these.
(Claims6The bacterium belonging to the genus Bacillus is Bacillus clarkey 7364 strain (FERM BP-7156)5The described DNA.
(Claims7Claim 1~ 6A recombinant plasmid containing the DNA according to any one of the above.
(Claims8Claim7A transformant transformed with the described plasmid.
(Claims9Claim7A transformant which is E. coli transformed with the described plasmid.
(Claims10Claim8Or9Culturing the transformant described in 1. and acting on starch, dextrin, amylopectin or amylose to produce mainly γ-cyclodextrin, the amount of β- and α-cyclodextrin produced is the same as the amount of γ-cyclodextrin produced A method for producing a protein, comprising collecting a protein having a cyclodextrin / glucanotransferase activity lower than that of the protein.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
SEQ ID NO: 2The amino acid sequences represented by amino acid numbers 1 to 28 are related to the signal peptide, and the amino acid sequences represented by amino acid numbers 29 to 702 are related to the protein of the enzyme (cyclodextrin / glucanotransferase). Therefore, the claims1The protein encoded by the DNA according to claim 1 includes a signal peptide and an enzyme (cyclodextrin / glucanotransferase) protein.2The protein encoded by the DNA described in is from only the enzyme proteinBecome.
[0013]
  The DNA according to claim 1 is:It has an amino acid sequence represented by amino acid numbers 1 to 702 of SEQ ID NO: 2 shown in the sequence listingIt is a DNA encoding a protein. Furthermore, the DNA of claim 2 is:It has an amino acid sequence represented by amino acid numbers 29 to 702 of SEQ ID NO: 2 shown in the sequence listingIt is a DNA encoding a protein.
[0014]
Claim3A DNA having a base sequence of 321 to 2426 bases of SEQ ID NO: 1 shown in the sequence listing described in the above, encodes an amino acid sequence represented by amino acid numbers 1 to 702 of SEQ ID NO: 2 in the sequence listing,3The DNA having the base sequence of 405 to 2426 bases of SEQ ID NO: 1 shown in the sequence listing in FIG. 5 encodes the amino acid sequence represented by amino acid numbers 29 to 702 of SEQ ID NO: 2 in the sequence listing. In addition, DNA which codes the amino acid sequence regarding a signal peptide has a base sequence of 321 to 404 bases of SEQ ID NO: 1 shown in the Sequence Listing.
[0015]
Claim4The DNA according to claim 1,~ 3Hybridizing with a DNA comprising a base sequence complementary to the DNA of any one of 1) under stringent conditions, and acting on starch, dextrin, amylopectin or amylose to produce mainly γ-cyclodextrin, The amount of β- and α-cyclodextrin produced is DNA encoding a protein having cyclodextrin / glucanotransferase activity which is lower than that of γ-cyclodextrin.
[0016]
The term “hybridization under stringent conditions” as used in the present invention means hybridization under conditions stronger than the Southern hybridization treatment conditions shown in the Examples. Specifically, the concentration of the constituent component is higher than that of the hybridization solution in the example, the hybridization temperature is high, the concentration of the constituent component of the cleaning solution is low, or the temperature of the cleaning solution is high. This is the case. In general, double-stranded DNA dissociates into single strands (denaturation) due to dissociation of hydrogen bonds by heat or alkali treatment, and gradually regenerates into double strand DNA by gradually lowering the temperature. In this denaturation regeneration, the higher the homology of the DNA double strand, the less the denaturation occurs and the easier the regeneration. Thus, when two different types of double-stranded DNA exist, they are denatured and then regenerated, so that different types of DNA form double-stranded DNA depending on the level of homology between the sequences. To do. Examining the homology between different DNAs by this method is called a hybridization method. The DNA of the present invention is complementary to a gene encoding an amino acid sequence represented by amino acid numbers 1 to 702 of SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence represented by amino acid numbers 29 to 702 of SEQ ID NO: 2. DNA having a base sequence that hybridizes with stringent DNA under stringent conditions (however, it acts on starch, dextrin, amylopectin or amylose to produce mainly γ-cyclodextrin, β- and α-cyclo The amount of dextrin produced is limited to DNA encoding a protein having low cyclodextrin / glucanotransferase activity compared to the amount of γ-cyclodextrin produced). If this is done under stringent conditions, DNA with high homology to the γ-CGTase structural gene can be selected efficiently, and it acts on starch, dextrin, amylopectin or amylose, and mainly acts as γ-cyclodextrin. The amount of β- and α-cyclodextrin produced is lower than that of γ-cyclodextrin, and the DNA encoding the protein having cyclodextrin / glucanotransferase activity is converted into the cyclodextrin / glucanotransferase. By appropriately selecting a protein having activity by a conventional method,4Can be obtained.
[0017]
Claims 1 to4The DNA described in (1) can be, for example, a DNA derived from a bacterium belonging to the genus Bacillus, and the bacterium belonging to the genus Bacillus can be the Bacillus clarkey 7364 strain (FERM BP-7156). However, claims 1 to4Are included in the present invention regardless of their origin.
[0018]
Furthermore, the present invention provides a method according to claim 1.6A recombinant plasmid containing the DNA according to any one of the above. From claim 16There is no restriction | limiting in particular in the vector for containing DNA of any one of these. The types of vectors and promoters will be described later. From claim 1 to the vector6The DNA described in any one of the above can be appropriately inserted by a known method.
[0019]
Furthermore, the present invention includes a transformant transformed with the above recombinant plasmid.
Examples of cells to be transformed include E. coli. Hosts other than E. coli will be described later. Transformation of the cells can be appropriately performed by a known method.
[0020]
In addition, the present invention mainly produces γ-cyclodextrin by acting on starch, dextrin, amylopectin or amylose, and the amount of β- and α-cyclodextrin produced is γ. A method for producing a protein comprising the step of collecting a protein having a cyclodextrin / glucanotransferase activity which is low compared to the amount of cyclodextrin produced; The method for culturing the transformant can be appropriately performed by a known method according to the type of the bacterial cell that is the source of the transformant. Proteins can also be collected using conventional methods. The collected protein can be appropriately subjected to purification or the like.
[0021]
In the first method for producing a CD composition of the present invention, a solution (raw material solution) containing at least one selected from the group consisting of starch, dextrin, amylopectin, and amylose is added to CGTase and β Cyclodextrin / glucanotransferase (specifically, CGTase produced by strains other than Bacillus clarkii species) has higher production activity of-and α-cyclodextrin than that of γ-cyclodextrin It reacts simultaneously and produces | generates (alpha) -CD, (beta) -CD, and (gamma) -CD.
[0022]
CGTase produced by strains other than Bacillus clarkii, which is a cyclodextrin / glucanotransferase, has higher β- and α-cyclodextrin production activity than γ-cyclodextrin production activity. To do.
In the present invention, CGTase produced by strains other than Bacillus clarkii species is CGTase that preferentially produces α- and / or β-CD compared to γ-CD. CGTases produced by strains other than Bacillus clarkii are cyclodextrin / glucanotransferase derived from the genus Bacillus, cyclodextrin / glucanotransferase derived from the genus Klebsiella, thermoanaero It can be at least one selected from the group consisting of a cyclodextrin / glucanotransferase derived from the genus Thremoanaerobactor and a cyclodextrin / glucanotransferase derived from the genus Brevibacterium. Examples of such CGTase include Bacillus sp. AL-6, Bacillus stearothermophilus, B. megaterium, and Bacillus cycle shown in Table 1 below. In addition to B. circulans and B. macerans, the following Table 1 shows B. ohbensis, Klebsiella pneumoniae, Thermoanaerobacter SP (Thermoana erobactor sp.), Brevibacterium sp (No. 9605), etc. can be mentioned.
[0023]
[Table 1]

[0024]
In the first method for producing a CD composition of the present invention, the concentration of the raw material solution is, for example, in the range of 1 to 30%, CGTase is used in an amount of 0.05 to 300 U (per 1 g of dry starch), and the pH range is 4.5 to 12 The composition containing α-CD, β-CD and γ-CD can be produced by performing an enzyme reaction at a temperature of 20 to 75 ° C. for 1 to 96 hours.
[0025]
In the second method for producing a CD composition of the present invention, CGTase which is the recombinant protein of the present invention is reacted with a raw material solution containing at least one selected from the group consisting of starch, dextrin, amylopectin and amylose, CGTases produced by strains other than Bacillus clarkii are reacted to produce α-CD, β-CD and γ-CD. The concentration of the raw material solution is, for example, in the range of 1 to 30%, 0.05 to 300 U (per 1 g of dried starch) of CGTase, which is the recombinant protein of the present invention, is added to the raw material solution, and the pH range of 4.5 to 12, 20 to 75 The enzyme reaction is performed at a temperature of 1 to 96 hours. Next, the reaction solution is heated to inactivate CGTase, which is the recombinant protein of the present invention, and then CGTase 0.05 to 300 U (per 1 g of dried starch) produced by a strain other than Bacillus clarkii species. In addition to the reaction solution, an enzyme reaction is carried out for 1 to 96 hours at a pH range of 4.5 to 12 and a temperature of 20 to 75 ° C. to produce a composition containing α-CD, β-CD and γ-CD. be able to.
[0026]
In the third method for producing a CD composition of the present invention, a strain other than Bacillus clarkii species is produced in a raw material solution containing at least one selected from the group consisting of starch, dextrin, amylopectin and amylose. CGTase is reacted, and then CGTase, which is the recombinant protein of the present invention, is reacted to produce α-CD, β-CD and γ-CD. The concentration of the raw material solution is, for example, in the range of 1 to 30%, and CGTase produced by a strain other than the Bacillus clarkii species is added to the raw material solution in an amount of 0.05 to 300 U (per 1 g of dried starch). The enzyme reaction is carried out for 1 to 96 hours at a pH range of 20 to 75 ° C. Next, the reaction solution is heated to inactivate CGTase produced by strains other than Bacillus clarkii species, and then the recombinant protein of the present invention, CGTase 0.05 to 300 U (per 1 g of dried starch). In addition to the reaction solution, an enzyme reaction is carried out for 1 to 96 hours at a pH range of 4.5 to 12 and a temperature of 20 to 75 ° C. to produce a composition containing α-CD, β-CD and γ-CD. be able to.
[0027]
The reaction conditions (enzyme use amount, reaction time, reaction temperature, etc.) are, for example, when the production amount of γ-cyclodextrin is 1, the production amount of α-cyclodextrin is in the range of 0.1 to 2, It can set so that the production amount of cyclodextrin may be in the range of 0.1-2. Preferably, when the production amount of γ-cyclodextrin is 1, the production amount of α-cyclodextrin is in the range of 0.1 to 1, and the production amount of β-cyclodextrin is in the range of 0.1 to 0.5. To do. The reaction conditions (enzyme use amount, reaction time, reaction temperature, etc.) are, for example, when the production amount of α-cyclodextrin is 1, the production amount of β-cyclodextrin is in the range of 0.1 to 1.5, It can also set so that the production amount of (gamma) -cyclodextrin may be in the range of 0.1-2. Preferably, when the production amount of α-cyclodextrin is 1, the production amount of β-cyclodextrin is in the range of 0.3 to 0.9 and the production amount of γ-cyclodextrin is in the range of 0.5 to 1. To do.
[0028]
For example, in the first method for producing a CD composition of the present invention, the ratio of the amount of CGTase produced by a strain other than Bacillus clarkii to the amount of CGTase that is the recombinant protein of the present invention is calculated. If the optimum temperature of CGTase varies depending on the type of CGTase, the production ratio is changed by changing the production ratio of each CD by bringing the reaction temperature close to the optimum temperature of one of the CGTases. be able to. In the second method for producing a CD composition of the present invention, the production conditions of CDs mainly composed of γ-cyclodextrin by CGTase which is the recombinant protein of the present invention (amount of enzyme used, reaction temperature, reaction time) and The production conditions of CDs mainly composed of α-cyclodextrin and / or β-cyclodextrin by CGTase produced by strains other than Bacillus clarkii species (enzyme use amount, reaction temperature, reaction time) and By changing the ratio, the ratio of the production amount can be changed.
In the third method for producing a CD composition of the present invention, the conditions for producing CDs containing α-cyclodextrin and / or β-cyclodextrin as a main component by CGTase produced by a strain other than Bacillus clarkii species (Enzyme use amount, reaction temperature, reaction time) and the production conditions of CDs mainly composed of γ-cyclodextrin by the recombinant protein of the present invention CGTase (enzyme use amount, reaction temperature, reaction time) and By changing the ratio, the ratio of the production amount can be changed.
[0029]
The solution containing a mixture of α-, β-, and γ-cyclodextrin obtained by the method for producing a CD composition of the present invention can be further purified to a cyclodextrin mixture syrup. This purification can be carried out by a conventional method similar to ordinary chickenpox or oligosaccharide syrup. For example, this purification can be performed by appropriately combining filtration for removing a solid content, decolorization by activated carbon treatment, and desalting by an ion exchange resin. Further, in the composition containing α-, β-, and γ-cyclodextrin obtained by the production method of the present invention, in addition to α-, β-, and γ-CD, monosaccharides such as glucose, various types In some cases, oligosaccharides (such as maltose) or dextrin may be contained, and these can be appropriately separated by a conventional method.
[0030]
Further, a solution containing a composition containing α-, β-, and γ-cyclodextrin obtained by the production method of the present invention was purified as described above to obtain a cyclodextrin mixture syrup, and then the obtained syrup Can be dried to obtain a cyclodextrin mixture powder. For drying syrup, a spray drying method or a freeze drying method can be used. Depending on the drying method, it can be in any form such as crystalline, lyophilized, powdered, granular or the like.
[0031]
The present invention is described in further detail below.
γ -CGTase gene
As described above, the γ-CGTase gene according to the present invention acts on a substrate selected from starch and its degradation products, and produces γ-cyclodextrin (γ-CD) as a main product. A DNA sequence encoding a protein having notransferase activity. Based on the partial amino acid sequence information of γ-CGTase, the enzyme can be obtained by PCR using the chromosomal DNA of the enzyme-producing bacterium (Bacillarsky strain 7364) as a template. It was determined by succeeding in obtaining a gene encoding, and also succeeding in expression in microorganisms such as Escherichia coli.
1) Deposit of microorganisms
E. coli JM109, which is transformed with a plasmid pGFT-01 containing a typical gene of the present invention (see Examples described later) and expresses a significant amount of the γ-CGTase of the present invention, is designated as GCG31. Deposited as FERM BP-7648 at the National Institute of Advanced Industrial Science and Technology.
2) γ -CGTase Enzymatic properties of
The present invention is based on the discovery of γ-CGTase by the present inventors, but this enzyme was produced from Bacillus claki 7364 strain, and its enzymological properties were as follows (implementation) Example 1). The γ-CD production activity was measured by the following method.
The reaction was started by keeping 450 μl of 1.5% soluble starch solution / 25 mM Gly-NaCl-NaOH buffer (pH 10.5) at 40 ° C. and adding 50 μl of an appropriately diluted enzyme solution. After 0, 5, 10, 15, 20, and 30 minutes from the start of the reaction, 500 μl of 0.05 N HCl was added to stop the reaction. A 5 mM BCG solution (in 20% Ethanol) was added to the reaction solution and mixed, and then kept at room temperature for 20 minutes. 2 ml of 1M BCG buffer solution (pH 4.2) was added, and the absorbance at 630 nm was measured. The γ-CD content in the reaction solution was determined using a calibration curve prepared in advance from the absorbance value. One unit of γ-CGTase cyclic activity was defined as the amount of enzyme that produced 1 μmol of γ-CD per unit time under the above reaction conditions.
a) The measurement of γ-CGTase cyclic activity by the BCG method was carried out using 150 mg of soluble starch in 10 ml of the prescribed pH buffer (pH 3-8: 1/4 × McIlvaine buffer (♦ in Fig. 1)), pH 8-10.5 : 25 mM Gly-NaCl-NaOH buffer (■ in Fig. 1), pH 10.5-11.9: 25 mM Na₂HPO_FourIt was dissolved in -NaOH buffer (■ in FIG. 1), and 450 μl thereof was used as a substrate solution. As a result, the optimum pH for cyclic activity by the BCG method was 10.0-10.5 (FIG. 1).
[0032]
b) Optimal temperature
For measurement of γ-CGTase cyclic activity by the BCG method, 150 mg of soluble starch was dissolved in 10 ml of 25 mM Gly-NaCl-NaOH buffer (pH 10.0), and 450 μl thereof was used as a substrate solution. The reaction was carried out at the prescribed temperature for 10 minutes. As a result, the optimum temperature for cyclic activity by the BCG method was 60 ° C. (FIG. 2).
c) pH stability
For the measurement of γ-CGTase cyclic activity by the BCG method, 150 mg of soluble starch was dissolved in 10 ml of 25 mM Gly-NaCl-NaOH buffer (pH 10.0), and 450 μl thereof was used as a substrate solution. Add 90 μl of the buffer solution with the specified pH to 10 μl of the enzyme (a) Use each pH buffer solution used to measure the optimum pH), leave it at 4 ° C for 24 hours, and then add 25 mM Gly-NaCl-NaOH buffer solution. (PH 10.0) 100 μl was added, and 50 μl was added to the substrate to carry out the reaction. The reaction was carried out at 40 ° C. for 20 minutes.
The pH stability was stable from pH 6 to 11 (FIG. 3).
[0033]
d) Temperature stability
For the measurement of γ-CGTase cyclic activity by the BCG method, 150 mg of soluble starch was dissolved in 10 ml of 25 mM Gly-NaCl-NaOH buffer (pH 10.0), and 450 μl thereof was used as a substrate solution. 90 μl of 25 mM Gly-NaCl-NaOH buffer (pH 10.0) was added to 10 μl of the enzyme, held at a predetermined temperature for 15 minutes, and then 50 μl was added to the substrate for reaction. The reaction was carried out at 40 ° C. for 10 minutes. This enzyme was treated at 50 ° C. for 15 minutes, and showed an activity of 80% or more without treatment (FIG. 4).
e) Molecular weight
The molecular weight determined by SDS-PAGE of this enzyme from the relative mobility with various standard proteins was 68 KDa.
[0034]
f) Isoelectric point
The isoelectric point determined from the relative mobility with various standard proteins by isoelectric focusing was 3.98.
g) N-terminal amino acid sequence
Analysis was performed using a gas phase protein sequencer “477A type” manufactured by Applied Biosystems by a conventional method. As a result, the enzyme had the amino acid sequence shown in SEQ ID NO: 3 at the N-terminus.
[0035]
h) Internal amino acid sequence
This enzyme was fragmented by a conventional method, and the N-terminal sequence of a peptide fragment isolated by HPLC was analyzed using a gas phase protein sequencer “477A type” manufactured by Applied Biosystems. It had an amino acid sequence.
[0036]
3) γ -CGTase Genetic analysis
Using a chromosomal DNA of Bacillus clrkii 7364 as a template, PCR reaction was performed using primers designed from the N-terminal amino acid sequence of the mature enzyme and the internal amino acid sequence. The obtained PCR fragment was found to be a 1470 bp gene fragment encoding the 9th N and subsequent genes from the N-terminus of γ-CGTase. Next, the antisense primer designed from the upstream sequence of the 1470 bp PCR fragment and the downstream using the circular DNA obtained by complete degradation of the chromosomal DNA with the restriction enzyme Sac I and then self-ligation as a template. Inverse PCR reaction was performed using the sense primer designed from the sequence, and the entire base sequence of the target enzyme gene was determined.
[0037]
4) Recombinant γ -CGTase Isolation and identification of genes
In order to confirm that the cloned DNA fragment is the gene encoding the target γ-CGTase, using Full F-primer: 5'-gACTTgTACTAAgACAACCTTACg-3 'and Full R-primer: 5'-gCATCggCTCTACTCATTTCA-3' PCR was performed using the chromosomal DNA as a template, and a 2530 bp PCR fragment containing the structural gene of γ-CGTase, a promoter and a transcription termination signal was obtained. The DNA fragment was then ligated into pGEM-T and inserted into E. coli JM-109. The transformed Escherichia coli (pGFT-01 / JM109) is cultured in LB medium for 16 hours, and then the cells are crushed and the insoluble material is removed by centrifugation. The supernatant is 0.35 U / kg with BCG and γ-CD cyclic activity. It was confirmed to have activity of ml. Furthermore, when this supernatant was used as a crude enzyme and allowed to act on 1% soluble starch (25 mM glycine-NaCl-NaOH buffer solution pH 10), it was confirmed by HPLC that γ-CD was the main product CGTase. confirmed.
Next, pGFT-01 / JM109 was cultured in large quantities (600 ml) in LB medium, and γ-CGTase was purified. Purification was performed in a two-step process using a γ-CD binding affinity column and gel filtration, and purified to a single band by SDS-PAGE.
The properties of the enzyme produced by this transformed Escherichia coli (pGFT-01 / JM109) are summarized in Table 2.
[0038]
[Table 2]

[0039]
The N-terminal sequence of γ-CGTase produced by this transformed Escherichia coli was analyzed by a conventional method using a gas phase protein sequencer “477A type” manufactured by Applied Biosystems. This enzyme was sequenced at the N-terminus. It had the amino acid sequence shown in No. 3.
The enzymological characteristics of this recombinant γ-CGTase were almost the same as those of γ-CGTase derived from Bacillus curkey 7364. Therefore, it was determined that the DNA encoding the above-mentioned recombinant γ-CGTase was derived from Bacillus clarkey 7364.
[0040]
5) Recombinant γ -CGTase Expression / production of genes encoding
a) Expression vector
A DNA molecule comprising a fragment encoding γ-CGTase according to the present invention in a host cell or in a state of being integrated into a chromosome and capable of expressing the same gene, in particular a host cell trait as a form of an expression vector If converted, γ-CGTase according to the present invention can be produced in the host cell. Therefore, according to the present invention, a DNA molecule, particularly an expression vector, comprising a gene encoding γ-CGTase according to the present invention is provided. Preferably the vector is a plasmid.
[0041]
The vector used in the present invention can be appropriately selected from viruses, plasmids, cosmid vectors and the like depending on the host cell to be used. For example, when the host cell is Bacillus subtilis, it is a pUB-type plasmid, and when E. coli is a λ phage-type bacteriophage, pBR322, BluesscriptIISK (+), pUC18, pUC19, pUC118, pUC119, pGEM-T pCR2.1, pLEX, Plasmid vectors such as pJL3, pSW1, pSE280, pSE420, pHY300PLK can be raised, but pBR322, BluesscriptIISK (+), pGEM-T, pUC18, pUC19, pUC118, pUC119, pCR2.1 are preferred for expression in E. coli. Yes, pHY300PLK is preferred for expression in Bacillus subtilis. Examples include pBR, pUC plasmids, and Yep, Ycp and YIP vectors in the case of yeast. This plasmid preferably contains a selection marker such as a drug resistance of the transformant or an auxotrophic marker. Furthermore, the expression vector preferably has a DNA sequence such as a promoter, terminator, ribosome binding site and transcription termination signal necessary for the expression of the γ-CGTase gene.
[0042]
As the promoter, commonly used promoters such as lac, trp, tac, and T7 commonly used in Escherichia coli can be preferably used, and expression is possible even if the promoter used for the expression of wild-type γ-CGTase is used as it is. Is possible. The sequence from No. 1 to No. 702 of the amino acid sequence shown in SEQ ID NO: 2 contains a signal peptide, but this sequence may be used as it is as shown in Examples below. In Bacillus subtilis, promoters such as xylose operon, subtilisin, and SPAC can be preferably used. In yeast, promoters such as ADH, PHO, GAL, and GAP can be preferably used.
[0043]
b) Transformant / culture
Examples of the host include Escherichia coli, Bacillus subtilis, actinomycetes, and yeast, and any host that does not produce amylase under the culture conditions of the transformant may be used. The method for producing CGTase according to the present invention comprises culturing the above-mentioned transformed cell, acting on a substrate selected from starch and its degradation product, and mainly using γ-cyclodextrin (γ-CD). A protein having cyclodextrin / glucanotransferase activity to be produced as a product is collected. The recombinant γ-CGTase according to the present invention is an expression product of the above gene. That is, γ-CGTase according to the present invention is an amino acid sequence represented by amino acid numbers 1 to 702 of SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence represented by amino acid numbers 29 to 702 of SEQ ID NO: 2 Sequence or an amino acid sequence in which one or more amino acid sequences thereof are substituted, deleted, inserted or added), and acts on, for example, starch dextrin, amylopectin or amylose, and is mainly composed of γ-cyclodextrin (γ-CD) Has a cyclodextrin / glucanotransferase activity that is produced as a product (ie, mainly produces γ-cyclodextrin, and the amount of β- and α-cyclodextrin is lower than that of γ-cyclodextrin) It is a protein.
[0044]
As a nutrient medium used for culturing the transformant of the present invention, any of a natural medium and a synthetic medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance, and, if necessary, a micronutrient required by the strain used. But you can. Examples of the carbon source include malto-oligosaccharides such as glucose and maltose, and hydrocarbons such as fructose, starch, dextrin, and glycerin. As the nitrogen source, amino chlorides such as ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, and glutamic acid, and inorganic nitrogen organic nitrogen compounds such as urea are used. Furthermore, nitrogen-containing organic natural products such as peptone, polypeptone, meat extract, yeast extract, CSL, soybean flour, soybean meal, dried yeast, casamino acid and the like can also be used. As the inorganic substance, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, zinc sulfate, sodium chloride, potassium chloride, calcium chloride and the like are used. In addition, micronutrients such as biotin and thiamine are used as necessary. The culture method is preferably a liquid culture method, and industrially, an aeration and agitation culture method is suitable. The culture temperature and pH may be selected under conditions that are most suitable for the growth of the transformant used. Although the culture time varies depending on the culture time, the culture is stopped when the production of recombinant γ-CGTase is confirmed, preferably when the production amount reaches the maximum. In order to collect the recombinant γ-CGTase of the present invention from the culture thus obtained, first, the cells in the culture solution are disrupted by a physical method such as ultrasonic waves, or an organic solvent, lysozyme, etc. After the cell membrane is lysed with the enzyme, the residue is removed by centrifugation or filtration. A concentrated crude enzyme solution for industrial use can be prepared by subjecting this to treatments such as ultrafiltration, salting out and solvent precipitation. Furthermore, the purified enzyme preparation can be easily obtained by combining this concentrated enzyme with well-known isolation and purification methods such as affinity chromatography with immobilized γ-CD, ion exchange chromatography and gel filtration chromatography. Can do.
[0045]
c) Method for producing a CD-containing composition having γ-cyclodextrin (γ-CD) and a desired CD balance (α-, β- and γ-CD balance)
According to the present invention, there is provided a method for producing a CD-containing composition having γ-cyclodextrin (γ-CD) and a desired CD balance (α-, β- and γ-CD balance) using the above-mentioned γ-CGTase. Provided. That is, in order to produce CD by the method of the present invention, for example, the present recombinant γ-CGTase enzyme solution (into an aqueous solution containing 1 to 30% starch (including starch or a composition fraction thereof, processed starch, etc.) ( Purified enzyme or crude enzyme) CGTase (α- and β-CGTase) produced by microorganisms from other sources alone or in combination with 0.1 to 300 U (per gram of dry starch), pH 4.5 to 12, temperature 20 to 60 ° C For 1 to 96 hours. Note that starch is used by heating in advance and liquefaction treatment as necessary.
[0046]
In addition to α-, β-, and γ-CD, etc., saccharides (containing composition) prepared by the method described above contain monosaccharides such as glucose, various oligosaccharides (such as maltose), or dextrin. May have. Further, if necessary, CD having a desired degree of single polymerization can be separated (by crystallization, chromatographic fractionation, fermentation treatment with yeast, enzyme treatment, etc.) and used. Needless to say, the form is an arbitrary form such as a crystalline form, a freeze-dried form, a powder form, and a granular form in addition to the contained composition form obtained by the above-described method. CDs according to the present invention are all foods and beverages that can be taken orally like CDs that have been marketed so far, for example, beverages such as teas and soft drinks, Japanese and Western confectionery such as candy, jelly and Japanese confectionery, yogurt, Add fragrances to dairy products such as ice cream, processed meat products such as ham and sausage, processed fishery products such as salmon, noodles, noodles, pickles, other cooked processed foods and instant foods, etc. By adding and coexisting as an emulsifying action and an excipient, and using CD, which is a safe food additive, the palatability and functionality of food and drink are improved extremely simply and effectively. Moreover, it can be used not only for foods and drinks but also for stabilizing and emulsifying active ingredients such as pharmaceuticals and cosmetics, and as excipients.
[0047]
The method of using the CD of the present invention is not particularly limited as long as the CD is present in foods, drinks, pharmaceuticals and cosmetics. For example, CD may be added simultaneously during the processing of the basic food or drink material, or after completion of the basic food or drink processing, and an addition method suitable for the actual state of the manufacturing process of various foods may be used. In the case of food and drink, the amount of CD added is not particularly limited as long as the original taste and flavor of the basic food and drink are not impaired, but it is generally preferred to add 20% by weight or less. If it is 20% by weight or more, there is a concern that the taste or taste of the basic food or drink may be changed due to the masking effect of the CD, so that the palatability may be reduced. In the case of pharmaceuticals and cosmetics, there is no particular limitation as long as the effective efficacy of the active ingredient is not impaired.
[0048]
【Example】
Examples of the present invention will be shown below, but this is for more specifically explaining the present invention, and the present invention is not limited to the scope of the following examples.
[0049]
This enzyme was produced from Bacillus claki 7364 strain and was prepared by the following method.
Neotac # 30T (manufactured by Nippon Shokuhin Kako Co., Ltd.) 1.0% (w / v) as a carbon source, Bacilscleraki 7364 (FERM BP-7156) as a carbon source, Soyaflower FT (Nisshin Oil) 0.5% as a nitrogen source, and yeast Extract (Difco) 0.5%, K₂HPO_Four 0.1% MgSO_Four・ 7H₂O 0.02% and Na₂CO_Three When cultured with shaking in a liquid medium containing 0.8% at 37 ° C for 48 hours, CGTase was secreted into the culture solution (blue value method 20 U / ml culture supernatant).
The obtained CGTase was purified by affinity chromatography.
[0050]
γ-CGTase activity was measured as γ-CD production activity (cyclization activity) by the following method. That is, 450 μl of 1.5% soluble starch solution / 25 mM Gly-NaCl-NaOH buffer (pH 10.5) was kept at 40 ° C., and the reaction was started by adding 50 μl of an appropriately diluted enzyme solution. After 0, 5, 10, 15, 20, and 30 minutes from the start of the reaction, 500 μl of 0.05 N HCl was added to stop the reaction. A 5 mM BCG solution (in 20% Ethanol) was added to the reaction solution and mixed, and then kept at room temperature for 20 minutes. 2 ml of 1M BCG buffer solution (pH 4.2) was added, and the absorbance at 630 nm was measured. The γ-CD content in the reaction solution was determined using a calibration curve prepared in advance from the absorbance value. One unit of γ-CGTase cyclic activity was defined as the amount of enzyme that produced 1 μmol of γ-CD per unit time under the above reaction conditions.
[0051]
Example 1 Enzymatic Chemical Properties of Purified γ-CGTase
Example 1-1 Preparation of purified enzyme
Cultivation of Bacillus claki 7364 strain and purification of γ-CGTase produced by the strain were performed by affinity chromatography.
[0052]
Example 1-2 Enzymatic properties of purified enzyme
1) Action
A soluble starch was used as a substrate, and a substrate solution was dissolved in 50 mM glycine-NaCl-NaOH (pH 10.0) buffer so as to be 10% (w / v). The purified enzyme was added to 5 ml of the substrate solution at 0.5 U / g DS and reacted at 50 ° C. for 48 hours. The reaction product was examined by the HPLC method described in Japanese Patent Application No. 2000-151056. After 48 hours, γ-CD was produced at 9.7%, and α- and β-CD were produced at 1.7 and 0.9% (HPLC area), respectively.
[0053]
2) Optimum pH
The measurement of γ-CGTase cyclic activity by the BCG method was performed by using 150 mg of soluble starch and 10 ml of a predetermined pH buffer (pH 3-8: 1/4 × McIlvaine buffer pH 8-10.5: 25 mM Gly-NaCl-NaOH buffer). Solution pH 10.5-11.9: 25 mM Na₂HPO_Four-NaOH buffer solution), and 450 μl thereof was used as a substrate solution. As a result, the optimum pH for cyclic activity by the BCG method was 10.0-10.5 (FIG. 1).
[0054]
3) Optimal temperature
For the measurement of γ-CGTase cyclic activity by the BCG method, 150 mg of soluble starch was dissolved in 10 ml of 25 mM Gly-NaCl-NaOH buffer (pH 10.0), and 450 μl thereof was used as a substrate solution. The reaction was carried out at the prescribed temperature for 10 minutes. As a result, the optimum temperature for cyclic activity by the BCG method was 60 ° C. (FIG. 2).
[0055]
4) pH stability
For the measurement of γ-CGTase cyclic activity by the BCG method, 150 mg of soluble starch was dissolved in 10 ml of 25 mM Gly-NaCl-NaOH buffer (pH 10.0), and 450 μl thereof was used as a substrate solution. Add 90 μl of the buffer solution with the specified pH to 10 μl of the enzyme (a) Use each pH buffer solution used to measure the optimum pH), leave it at 4 ° C for 24 hours, and then add 25 mM Gly-NaCl-NaOH buffer solution. (PH 10.0) 100 μl was added, and 50 μl was added to the substrate to carry out the reaction. The reaction was carried out at 40 ° C. for 20 minutes.
The pH stability was stable from pH 6 to 11 (FIG. 3).
[0056]
5) Temperature stability
For the measurement of γ-CGTase cyclic activity by the BCG method, 150 mg of soluble starch was dissolved in 10 ml of 25 mM Gly-NaCl-NaOH buffer (pH 10.0), and 450 μl thereof was used as a substrate solution. 90 μl of 25 mM Gly-NaCl-NaOH buffer (pH 10.0) was added to 10 μl of the enzyme, held at a predetermined temperature for 15 minutes, and then 50 μl was added to the substrate for reaction. The reaction was carried out at 40 ° C. for 10 minutes. This enzyme was treated at 50 ° C. for 15 minutes, and showed an activity of 80% or more without treatment (FIG. 4).
[0057]
6) Molecular weight
The molecular weight determined by SDS-PAGE of this enzyme from the relative mobility with various standard proteins was 68 KDa.
[0058]
7) Isoelectric point
The isoelectric point determined from the relative mobility with various standard proteins by isoelectric focusing was 3.98.
[0059]
8) N-terminal amino acid sequence
Analysis was performed using a gas phase protein sequencer “477A type” manufactured by Applied Biosystems by a conventional method. As a result, the enzyme had the amino acid sequence shown in SEQ ID NO: 3 at the N-terminus.
[0060]
9) Internal amino acid sequence
This enzyme was fragmented by a conventional method, and the N-terminal sequence of a peptide fragment isolated by HPLC was analyzed using a gas phase protein sequencer “477A type” manufactured by Applied Biosystems. It had an amino acid sequence.
[0061]
(Example 2)
Example 2-1 Preparation of chromosomal DNA
The chromosomal DNA of Bacillus clarkii 7364 has been reported by N. Declerck et al. (N. Declerck, P. Joyet, D. Le Coq and H. Heslot, J. Biotech., 8, 1998, 23-38). For reference, it was prepared as follows. Bacterial cells were collected by centrifugation (8,000 rpm, 10 min) from a culture solution (40 ml) obtained by culturing Bacillus clarkii 7364 strain in an enzyme production medium. The resulting cells were washed twice with 5 ml of TESS buffer (30 mM Tris / HCl (pH 7.5), 5 mM EDTA, 50 mM NaCl, 25% sucrose) and suspended in 3 ml of the same buffer. did. The cell suspension was treated at 65 ° C. for 10 minutes and cooled to 37 ° C. 1 ml of an aqueous lysozyme solution (50 mg / ml) was added and reacted at 37 ° C. for 1 hour. Subsequently, proteinase K was dissolved in 25 mg / ml of water, self-digested at 37 ° C. for 1 hour, 500 μl thereof was added, and reacted at the same temperature for 2 hours. The reaction was stopped by adding 1 ml of 10% SDS and treating at 65 ° C for 10 minutes. Treat the reaction solution with TE-saturated phenol and phenol / chloroform (twice), dissolve the chromosomal DNA recovered by ethanol precipitation in 1.2 ml of TE, and then add the RNAase solution (500 μg / ml) to 10 μg / ml. And reacted at 37 ° C. for 2 hours. Chromosomal DNA was collected by ethanol precipitation after treatment with phenol / chloroform (twice). By this operation, 162 μg of chromosomal DNA was obtained.
[0062]
Example 2-2 Acquisition of DNA fragment encoding γ-CGTase and determination of nucleotide sequence
Preparation of plasmids, restriction digestion, ligation, and transformation of E. coli were performed according to previously reported methods (Sambrook, J., Fritsch, EF & Maniatis, T. (1982) Molecular cloning: a laboratory manual, 2^nd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The base sequence was determined by the dideoxy chain termination method using a multicapillary DNA analysis system CEQ2000 manufactured by Beckman Coulter. Determination of the base sequence of the DNA strand and computer analysis were performed using GENETYX-WIN.
A partial fragment of the gene encoding γ-CGTase should be subjected to PCR using chromosomal DNA as a template and N-terminal F primer: 5'-AAYgTIAAITAYgCIgARgARgT-3 'and Domain Cr primer: 5'-gCRTCICCIggYTTICCCATDCC-3'. It was prepared with. The N-terminal F primer is the N-terminal amino acid sequence of the mature protein determined by Edman's method, the 9th and subsequent NVNYAEE, and the Domain Cr primer is a peptide fragment obtained by random digestion of the mature protein with protease. A part of the N-terminal amino acid sequence was designed based on PMGKPGDA. The PCR reaction conditions are summarized below.
[0063]
[Table 3]

[0064]
The reaction solution was heated at 94 ° C. for 5 minutes, and then a cycle of 94 ° C. for 1 minute, 51 ° C. for 2 minutes, and 72 ° C. for 3 minutes was repeated 30 times. Finally, the mixture was incubated at 72 ° C. for 10 minutes. The about 1500 bp fragment obtained by this PCR was ligated to T-vector pGEM-T, and the nucleotide sequence was determined. In the amino acid sequence deduced from the base sequence, a sequence that completely matches the N-terminal amino acid sequence obtained from Edman analysis (the mature enzyme N-terminal amino acid sequence and the enzyme internal peptide N-terminal sequence) was found, and further amylase A consensus sequence of family enzymes was found. Therefore, the gene fragment was determined to be a 1470 bp fragment encoding the 9th and subsequent fragments from the N-terminus of the γ-CGTase mature enzyme protein.
[0065]
The chromosomal DNA prepared in the above example was completely digested with restriction enzymes Sac I, EcoRI, BamHI, etc. and separated by agarose gel electrophoresis. In the PCR performed when the separated DNA fragment was transferred to a nylon membrane by a conventional method to obtain the above 1470 bp fragment, PCR was carried out under the same conditions by confusion with dNTP and dig-labeled dNTP at a ratio of 1: 1. The Dig-labeled 1470 bp DNA fragment prepared in this way was Southern hybridized with the DNA fragment immobilized on the nylon membrane (Southern et al, J. Mol. Biol., 98, 503-517, 1975). Conditions are 5 × SSC, 1% SDS, 10% dextran, 0.5 mg / ml In a solution consisting of salmon denatured DNA, hybridization is performed at 65 ° C. for 16-20 hours, and then 2 × SSC, 1% SDS. The solution was washed 3 times at 65 ° C. for 30 minutes. As a result, a DNA fragment of about 2500 bp digested with SacI hybridized.
[0066]
The following experiment was then performed to obtain the 5′- and 3′-upstream and downstream regions of the fragment. First, after complete digestion of chromosomal DNA with Sac I, T4DNA ligase was added under appropriate conditions so that monomeric circles were formed, and the digestion was allowed to react at 16 ° C. overnight (1 μg of digested chromosomal DNA, 10 x ligation buffer 10 μl, T4 ligase 700 U / 1 ml). Using the obtained Sac I-self-circulized DNA molecule as a template, PCR was performed using inverse anti primer: 5′-gATCTgTTACAATATgATAAAT-3 ′ and inverse sense primer: 5′-TTATTAGACggTCAATCGTTA-3 ′. These primers were designed based on the 5′-upstream region of the aforementioned 1470 bp fragment for the inverse anti primer and the 1470 bp fragment 3′-downstream for the inverse sense primer. The PCR reaction conditions are summarized below.
[0067]
[Table 4]

[0068]
The above reaction mixture was heated at 94 ° C for 5 minutes, then cycled at 94 ° C for 0.5 minute, 47 ° C for 0.5 minute, and 72 ° C for 0.5 minute 10 times, 94 ° C for 0.5 minute, 50 ° C for 0.5 minute, 72 ° C A cycle of 10 minutes at 94 ° C, 10 minutes at 94 ° C, 1 minute at 50 ° C, 2 minutes at 50 ° C, and 3 minutes at 72 ° C were repeated 10 times. The fragment of about 2000 bp obtained by this PCR was ligated to T-vector pGEM-T, and the nucleotide sequence was determined. As a result, about 0.4 Kbp upstream base sequence including the remaining N-terminal sequence and start codon of γ-CGTase, initiation codon, ribosome binding region (RBS) and promoter sequence, and the remaining C-terminal region and terminal codon, including inverted repeat. The base sequence in the downstream region of 0.2 Kbp was determined, and the base sequence shown in SEQ ID NO: 1 was determined.
[0069]
Example 2-3 Transformation of recombinant plasmid pGFT-01 and E. coli
Full F-primer: 5'-gACTTgTACTAAgACAACCTTACg-3 'and Full R-primer: 5'-gCATCggCTCTACTCATTTCA-3 using the γ-CGTase gene shown in SEQ ID NO: 1 and its flanking region as a template for chromosomal DNA PCR was performed using 'as a primer. The primer used in the reaction was designed from a base sequence 237 bp upstream from the start codon and 77 bp downstream from the stop codon.
The PCR reaction conditions are summarized below.
[0070]
[Table 5]

[0071]
The reaction solution was heated at 94 ° C. for 5 minutes, and then a cycle of 94 ° C. for 1.0 minute, 55 ° C. for 1.5 minutes, and 72 ° C. for 3 minutes was repeated 32 times, and finally the temperature was kept at 72 ° C. for 10 minutes.
After ligation of the obtained DNA fragment of about 2.5 Kbp into T-vector pGEM-T and insertion into E. coli JM109, a large amount of plasmid pGFT-01) was prepared, and the entire nucleotide sequence was confirmed by the primer walking method. It was confirmed that DNA having at least the nucleotide sequence shown in SEQ ID NO: 1 was contained.
[0072]
Example 3 Purification and enzymological properties of γ-CGTase produced by E. coli
pGFT-01 / JM109 was cultured in large amounts (600 ml) in LB medium, and γ-CGTase was purified. According to the method of Example 1 above, purification can be performed by a two-step process using a γ-CD binding affinity column and gel filtration, and the product was purified to a single band by SDS-PAGE. 15.5 mg (79.8 U) of protein was obtained (73% recovery).
The properties of the enzyme produced by this transformed Escherichia coli (pGFT-01 / JM109) are summarized in Table 6.
[0073]
[Table 6]

FIG. 5 shows the results of SDS-PAGE of the enzyme (1) produced by B. clarkii and the enzyme produced by E. coli (2). The properties of the E. coli-producing enzyme showed almost the same properties as the enzyme produced by B. clarkii.
[0074]
(Example 4) Preparation of CD-containing composition using γ-CGTase produced by E. coli (1)
Corn starch was liquefied using α-amylase by a conventional method to prepare a starch liquefaction solution having a concentration of 20% by weight and a glucose equivalent of 7. Next, this starch liquefaction solution was adjusted to pH 7, and then the E. coli produced γ-CGTase described in Example 3 was concentrated using a UF concentration membrane (PM-10) as a crude enzyme solution, and 1 unit / g substrate was added. And reacted at 55 ° C. for 48 hours. Thereafter, the reaction solution was heated to inactivate the enzyme, and purified such as decolorization and ion exchange to prepare a containing composition containing CD. The sugar composition was determined by HPLC method.
[0075]
[Table 7]

[0076]
(Example 5) Preparation of CD-containing composition using γ-CGTase produced by Escherichia coli (2)
Corn starch was liquefied using α-amylase by a conventional method to prepare a starch liquefaction solution having a concentration of 10% by weight and a glucose equivalent of 7. Next, this starch liquefaction solution was adjusted to pH 7, and then 2 units / g substrate was added as a crude enzyme solution obtained by concentrating the Escherichia coli-produced γ-CGTase described in Example 3 using a UF concentration membrane (PM-10). And reacted at 55 ° C. for 48 hours. Thereafter, the reaction solution was heated to 90 ° C. to inactivate the enzyme, cooled to 75 ° C., added with 40 units / g substrate of CGTase derived from Bacillus genus (manufactured by Nippon Shokuhin Kako Co., Ltd.), and reacted for 24 hours. . The composition containing CD was prepared by heating to 90 ° C. to inactivate the enzyme, purifying it such as decolorization and ion exchange. The sugar composition was determined by HPLC method.
[0077]
[Table 8]

[0078]
(Example 6)
Corn starch was liquefied using α-amylase by a conventional method to prepare a starch liquefaction solution having a concentration of 10% by weight and a glucose equivalent of 7. Next, this starch liquefaction solution was adjusted to pH 7, and then the E. coli-produced γ-CGTase described in Example 3 was concentrated using a UF concentration membrane (PM-10) as a crude enzyme solution, 3 units / g substrate, and CGTase derived from Bacillus genus (manufactured by Nippon Shokuhin Kako Co., Ltd.) with 20 units / g substrate was added at the same time and reacted at 55 ° C. for 72 hours. Thereafter, the reaction solution was heated to 90 ° C. to deactivate the enzyme, and purified such as decolorization and ion exchange to prepare a containing composition containing CD. The sugar composition was determined by HPLC method.
[0079]
[Table 9]

[0080]
【The invention's effect】
The present invention is a recombinant γ-CGTase-producing microorganism produced by genetic engineering techniques based on the alkalophilic Bacillus genus bacteria found by the present inventors, in particular, the γ-CGTase gene derived from Bacillus clarkey 7364. If this is cultured, it is extremely excellent in that the productivity of the enzyme can be improved and γ-CGTase with less impurities can be produced. In addition, as a result of making it possible to produce this recombinant enzyme on an industrial scale, CD having γ-cyclodextrin (γ-CD) and desired CD balance (α-, β- and γ-CD balance) It is also very valuable in that the production of the containing composition can be made remarkably efficiently.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1. Effect of pH on γ-CGTase cyclic activity by BCG method.
[Fig. 2] Effect of temperature on γ-CGTase cyclic activity by BCG method.
FIG. 3 shows pH stability against γ-CGTase cyclic activity by BCG method.
FIG. 4 shows temperature stability against γ-CGTase cyclic activity by BCG method.
FIG. 5 shows the results of SDS-PAGE of an enzyme produced by B. clarkii and an enzyme produced by Escherichia coli.

Claims (10)

 DNA encoding a protein having an amino acid sequence represented by amino acid numbers 1 to 702 of SEQ ID NO: 2 shown in the sequence listing. DNA encoding a protein having an amino acid sequence represented by amino acid numbers 29 to 702 of SEQ ID NO: 2 shown in the sequence listing. DNA having a base sequence of 321 to 2426 bases or a base sequence of 405 to 2426 bases of SEQ ID NO: 1 shown in the Sequence Listing. It hybridizes under stringent conditions with DNA comprising a base sequence complementary to the DNA according to any one of claims 1 to 3 , and acts on starch, dextrin, amylopectin or amylose to produce mainly γ-cyclo A DNA encoding a protein that produces dextrin and has a low cyclodextrin / glucanotransferase activity in which the amount of β- and α-cyclodextrin is lower than that of γ-cyclodextrin. The DNA according to any one of claims 1 to 4 , wherein the DNA is derived from a Bacillus bacterium. The DNA according to claim 5, wherein the bacterium belonging to the genus Bacillus is Bacillus clarkey 7364 strain (FERM BP-7156). A recombinant plasmid containing the DNA according to any one of claims 1 to 6 . A transformant transformed with the plasmid according to claim 7 . A transformant which is Escherichia coli transformed with the plasmid according to claim 7 . The transformant according to claim 8 or 9 is cultured and acts on starch, dextrin, amylopectin or amylose to mainly produce γ-cyclodextrin, and the amount of β- and α-cyclodextrin produced is γ-cyclodone. A method for producing a protein, comprising collecting a protein having a cyclodextrin / glucanotransferase activity lower than the amount of dextrin produced.

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