JP4370395B2 - Tannase, its gene and method for producing tannase - Google Patents

Description

【００２１】
本発明のタンナーゼは本明細書記載の理化学的性質を有する限り、その由来により限定されるものではないが、好ましくは微生物由来の蛋白質であり、更に好ましくは糸状菌由来の蛋白質であり、更に好ましくはアスペルギルス属に属する糸状菌由来の蛋白質であり、最も好ましくは黄麹菌（アスペルギルス・オリゼ、アスペルギルス・ソーヤ、アスペルギルス・タマリ）由来の蛋白質である。更に、本発明のタンナーゼは遺伝子組換え技術により得られるタンナーゼも含むものである。
【００２２】
特定の実施形態においては、本発明のタンナーゼ活性を有する蛋白質は配列番号１に表されるアミノ酸配列からなる蛋白質である。しかしながら、本発明のタンナーゼ活性を有する蛋白質はタンナーゼ活性を有する限り、配列番号１で表されるアミノ酸配列からなる蛋白質に限定されるものではない。すなわち、本発明のタンナーゼ活性を有する蛋白質は、配列番号１で表されるアミノ酸配列において１若しくは複数（好ましくは２〜５０個、より好ましくは２〜１０個、より好ましくは数個）のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつタンナーゼ活性を有する蛋白質であり得る。ここで、「１もしくは複数のアミノ酸が付加、欠失もしくは置換されたアミノ酸配列」とあるのは、目的とするタンナーゼ活性が得られる限り、配列番号１で表されるアミノ酸配列が、該アミノ酸配列とは異なる配列に付加されてもよく、又、該アミノ酸配列の一部が欠失又は置換されてもよいことを意味する。例えば、配列番号１で表されるアミノ酸配列の第１番目のメチオニンが欠失しても、本発明の目的とする熱安定性に優れ、高温・中性域で良く反応するタンナーゼ活性が得られる限り、かかる欠失した配列も本発明に含まれることを意味する。
【００２３】
また、本発明のタンナーゼ活性を有する蛋白質は、配列番号１で表わされる全アミノ酸配列の30％以上を占める連続するアミノ酸配列領域と65％以上、好ましくは70％以上、更に好ましくは80％以上の配列相同性を示すアミノ酸配列からなる蛋白質又はその部分断片であり、かつタンナーゼ活性を有する蛋白質であり得る。更には、本発明のタンナーゼ活性を有する蛋白質は、配列番号１で表わされる全アミノ酸配列の60％以上を占める連続するアミノ酸配列領域と65％以上、好ましくは70％以上、さらに好ましくは80％以上の配列相同性を示すアミノ酸配列からなる蛋白質又はその部分断片であり、かつタンナーゼ活性を有する蛋白質であり得る。
【００２４】
更に、本発明のタンナーゼ活性を有する蛋白質は、配列番号１で表わされる全アミノ酸配列の90％以上を占める連続するアミノ酸配列領域と65％以上、好ましくは70％以上、更に好ましくは80％以上の配列相同性を示すアミノ酸配列からなる蛋白質又はその部分断片であり、かつタンナーゼ活性を有する蛋白質であり得る。
次に、本酵素の力価の測定法について説明する。本酵素の活性測定は、下記の測定法１に従って行なった。尚、30℃においてタンニン酸中のエステル結合を、１分間に１μmol加水分解する酵素量を、１単位（Ｕ）とする。
【００２５】
〔測定法１〕アグリカルチュアル・バイオロジカル・ケミストリー（31巻、513頁〜518頁、1967年）に記載の方法を一部改変して本酵素の力価を測定する。すなわち、タンニン酸（ジ没食子酸）のエステル結合が加水分解される度合を、310nmの吸光度減少を分光光学的に測定することにより算出する。
【００２６】
具体的には、1.0 ml の基質溶液〔0.35％ タンニン酸(ジ没食子酸)を含有する50mM クエン酸緩衝液（ｐＨ5.5）〕を30℃、5分間平衡化し、適宜希釈したタンナーゼ溶液0.25mlを加え、30℃、15分間反応させる。次いで5.0mlの90％エタノールを混合して反応を停止後、この溶液を0.25ml採取して別の試験管に移し、5.0mlの90％エタノールを混合して希釈を行なう。次いで310nm における吸光度変化を、分光光度計（U-2001, 日立社製）を用いて測定する。酵素の代わりに緩衝液を加えて同様に測定したものをブランク値とし、ブランク値との測定値の差を酵素反応量とする。
【００２７】
〔２〕本発明の新規タンナーゼ遺伝子
特定の実施形態においては、本発明のタンナーゼ遺伝子は上記理化学的性質を有する蛋白質をコードするタンナーゼ遺伝子であり得る。
更には、特定の実施形態においては、本発明のタンナーゼ遺伝子は配列番号２で表される塩基配列からなるタンナーゼ遺伝子であり得る。しかしながら、タンナーゼ活性を有する蛋白質をコードする限り、配列番号２で表される塩基配列からなるタンナーゼ遺伝子に限定されるものではない。すなわち、本発明のタンナーゼ遺伝子は、配列番号２で表される塩基配列からなるＤＮＡと相補的な塩基配列からなるＤＮＡとストリンジェントな条件下でハイブリダイズし、かつタンナーゼ活性を有する蛋白質をコードするタンナーゼ遺伝子であり得る。
【００２８】
ストリンジェントな条件とは、特異的なハイブリッドのシグナルが非特異的なハイブリッドのシグナルと明確に識別される条件であり、使用するハイブリダイゼーションの系と、プローブの種類、配列及び長さによって異なる。そのような条件は、ハイブリダイゼーションの温度を変えること、洗浄の温度及び塩濃度を変えることにより決定可能である。例えば、非特異的なハイブリッドのシグナルまで強く検出されてしまう場合には、ハイブリダイゼーション及び洗浄の温度を上げると共に、必要により洗浄の塩濃度を下げることにより特異性を上げることができる。また、特異的なハイブリッドのシグナルも検出されない場合には、ハイブリダイゼーション及び洗浄の温度を下げると共に、必要により洗浄の塩濃度を上げることにより、ハイブリッドを安定化させることができる。このような最適化は、本技術分野の研究者が容易に行ない得るものである。
【００２９】
ストリンジェントな条件の具体例としては、例えば、プローブとしてDNAプローブを用い、ハイブリダイゼーションは、5 ×SSC 、1.0 %(W/V)核酸ハイブリダイゼーション用ブロッキング試薬（ベーリンガ・マンハイム社製）、0.1 %(W/V)N-ラウロイルサルコシン、0.02 %(W/V)SDSを用い一晩（８〜１６時間程度）で行なう。洗浄は、0.1〜0.5×SSC、0.1%(W/V)SDS、好ましくは0.1×SSC 、0.1%(W/V)SDSを用い、15分間、2回行なう。ハイブリダイゼーション及び洗浄を行なう温度は65℃以上、好ましくは68℃以上である。
【００３０】
また、本発明のタンナーゼ遺伝子は、配列番号２で表される塩基配列からなるＤＮＡと70%以上、好ましくは80%以上、更に好ましくは90%以上の配列相同性を示し、かつタンナーゼの機能を有する蛋白質をコードするタンナーゼ遺伝子であり得る。
【００３１】
上記のアミノ酸配列又は塩基配列の解析において、２つのアミノ酸配列又は塩基配列における配列相同性を決定するために、先ず配列は、比較に最適な状態に前処理される。例えば、一方の配列にギャップを入れることにより、他方の配列とのアラインメントの最適化を行なう。その後、各部位におけるアミノ酸残基又は塩基が比較される。第一の配列における、ある部位に、第二の配列の相当する部位と同じアミノ酸残基又は塩基が存在する場合、それらの配列は、その部位において同一である。
２つの配列における配列相同性は、配列間での同一である部位数の全部位（全アミノ酸又は全塩基）数に対する百分率で示される。
【００３２】
上記の原理に従い、２つのアミノ酸配列又は塩基配列における配列相同性は、Karlin 及び Altschulのアルゴリズム(Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990及びProc. Natl. Acad. Sci. USA 90:5873-5877, 1993)により決定される。このようなアルゴリズムを用いたBLASTプログラムがAltschul等によって開発された(J. Mol. Biol. 215:403-410, 1990)。更に、Gapped BLASTはBLASTより感度よく配列相同性を決定するプログラムである(Nucleic Acids Res. 25:3389-3402, 1997)。上記のプログラムは、主に与えられた配列に対し、高い配列相同性を示す配列をデータベース中から検索するために用いられる。これらは、例えば、米国National Center for Biotechnology Informationのインターネット上のウェブサイトにおいて利用可能である。
【００３３】
ただし、上記BLASTソフトウェアで有意な配列相同性を示す配列が見つからない場合には、更に高感度なFASTAソフトウェア（W.R. Pearson and D.J. Lipman,Proc. Natl. Acad. Sci., 85:2444-2448, 1988）を用いて配列相同性を示す配列をデータベースから検索することもできる。FASTAソフトウェアは、例えば、ゲノムネットのウェブサイトで利用できる。この場合も、パラメーターは、デフォルト値を用いる。例えば、塩基配列についての検索を行なう場合は、データベースにnr-ntを用い、ktup値は6を用いる。
【００３４】
上記の各方法は、主にデータベース中から配列相同性を示す配列を検索するために用いられるが、個別の配列の配列相同性を決定する手段として、本発明では、Genetyx Mac Ver（トウエア開発社）のホモロジー解析を用いることができる。この方法は、高速、かつ高感度な方法として多用されているLipman-Pearson法(Science, 227:1435-1441, 1985)に基づくものである。塩基配列の配列相同性を解析する際は、可能であれば蛋白質をコードしている領域（CDS又はORF）を用いる。パラメーターとしては、Unit Size to compare = 2 , Pick up Location = 5とし、結果を％表示させる。最も高いポイントを示したアラインメントの配列相同性を結果として用いるが、問い合わせ配列の30%以上、50%以上、又は70%以上のオーバーラップを示さない場合は、機能的に相関しているとは必ずしも推定されないため、２つの配列間の配列相同性を示す値としては用いない。例えば、数塩基／残基程度の完全一致領域があったとしても、それは偶然の結果に過ぎない可能性が高く、また、全体の数％程度の長さにおける一致では、特定の機能モチーフが含まれていたとしても、全体として同じ機能を果たすと考えることは困難である。
【００３５】
〔３〕微生物を用いた本酵素の生産
本酵素を製造するために使用される微生物としては、本酵素の生産能を有する限り、遺伝子組換えの有無に関わらず、いかなる微生物を使用してもよく、好ましくは糸状菌、更に好ましくはアスペルギルス属に属する糸状菌が使用される。具体的には、例えば、アスペルギルス・オリゼ RIB40(ATCC 42149)等を挙げることができる。また、該菌株の変異株を用いることも可能である。該菌株の変異株を得る方法としては、例えば、ラジオアイソトープ、紫外線、ニトロソグアニジン等を用いて原株に突然変異を起こさせる方法を用いることができる。
【００３６】
当然のことながら、本酵素の生産に使用する微生物は、本酵素の生産能を有すると同時に、例えば従来のタンナーゼの生産能を兼ね備えたものであってもよい。そのような微生物としては、本来両方のタンナーゼの生産能を兼ね備えたものや、本来ある種のタンナーゼの生産能を有する微生物に、別種のタンナーゼをコードする遺伝子を導入して複数種のタンナーゼの生産能を兼ね備えさせたものが含まれる。複数種のタンナーゼの生産能を兼ね備えた微生物を、各種の培養条件下で培養することによって、目的に応じ、各タンナーゼの生産比率を任意に変化させて酵素生産させることも可能である。
【００３７】
上記の微生物を培地に培養し、得られる培養物から本酵素を採取する。培養法は、通常の固体培養法でもよく、液体培養法を採用することが好ましい。培地は、微生物を培養する通常の培地、すなわち炭素源、窒素源、無機物、その他の栄養素を適切な割合で含有するものであれば、合成培地又は天然培地のいずれでも使用できる。
【００３８】
炭素源としては、資化可能な炭素化合物、例えば、グルコース、マルトース、デンプン加水分解物、グリセリン、フラクトース、糖蜜等が使用される。又、窒素源としては、利用可能な窒素化合物、例えば、酵母エキス、ペプトン、ソイトン、肉エキス、コーンスチープリカー、大豆粉、大豆もしくは小麦ふすま侵出液、アミノ酸、硫安、硝酸アンモニウム等が使用される。その他、食塩、塩化カリウム、硫酸マグネシウム、塩化マンガン、硫酸第一鉄、リン酸第一カリウム、リン酸第二カリウム、炭酸ナトリウム、酢酸ナトリウム等の種々の塩類、ビタミン類、消泡剤等が使用される。これらの培地成分は、単独で、又は適宜組み合わせて用いることができる。
【００３９】
微生物の培養は、使用する微生物の性質及び培地の形状に応じて通気撹拌培養、振盪培養、静置培養等を適宜選択し、培地の初発ｐＨを６〜７程度に調整した後、25〜35℃、好ましくは30℃前後で24〜140時間、好ましくは60〜100時間行えばよい。培養終了後、培養物より本酵素を採取するためには、通常の酵素採取手段を用いることができる。すなわち、酵素が菌体外部に放出される場合には、濾過又は遠心分離等により菌体を分離して、酵素液を含む培養液を回収することができる。また酵素が菌体内部に存在する場合には、超音波破砕機、フレンチプレス、リゾチーム等の細胞壁溶解酵素、界面活性剤等を用いて、濾過又は遠心分離等により分離した菌体から酵素を抽出し、次いで、濾過又は遠心分離等を用いて不溶物を除去することにより、本酵素を含有する粗酵素液を得ることができる。
【００４０】
このようにして得られた粗酵素液から、本酵素を更に精製するには、通常の蛋白質精製法を使用することができる。具体的には、例えば、硫安塩析法、有機溶媒沈澱法、イオン交換クロマトグラフィー、疎水クロマトグラフィー、ゲル濾過クロマトグラフィー、吸着クロマトグラフィー、アフィニティークロマトグラフィー、電気泳動法等を単独で、又は適宜組み合わせて用いることができる。
【００４１】
〔４〕遺伝子工学的手法による本酵素遺伝子のクローニング及び本酵素の生産
以下に、本をコードする遺伝子（以下、文脈により「tan遺伝子」という）を含むＤＮＡが挿入された組み換え体ＤＮＡにより宿主細胞を形質転換又は形質導入し、得られた組み換え微生物を用いて本酵素を生産する方法について詳細に説明する。
【００４２】
１．tan遺伝子のクローニング
tan遺伝子を含むＤＮＡは、染色体ＤＮＡ、又はｃＤＮＡ由来の野生型遺伝子をクローニングすることにより得られる。又、染色体ＤＮＡ又はｃＤＮＡを鋳型としてポリメラーゼ連鎖反応（以下「ＰＣＲ」という）を行なうことにより該遺伝子を増幅することもできる。更には化学合成法を用いて該遺伝子を構築することも可能である。以下、アスペルギルス属に属する微生物の染色体ＤＮＡを鋳型にPCRによりtan遺伝子をクローニングする方法を例にとり、tan遺伝子を含むＤＮＡの単離法について説明する。
【００４３】
PCR法によるtan遺伝子の増幅に鋳型ＤＮＡとして用いる染色体ＤＮＡは、本発明のタンナーゼ遺伝子を有する微生物由来であればいかなる微生物由来の染色体ＤＮＡでも用いることができる。これらの微生物として、例えば、アスペルギルス・オリゼRIB40(ATCC 42149)株等が挙げられる。プライマーとしては、tan遺伝子を含むDNA断片の増幅が可能であればいかなる配列のプライマーを用いてもよい。その例としては、配列番号３、４で表されるプライマー等が挙げられる。
【００４４】
PCR反応に用いるＤＮＡポリメラーゼとしては、例えば、TaKaRa Ex Taq（タカラバイオ株式会社製）、KOD DNA Polymerase（東洋紡績株式会社製）等を用いることが可能であるが、PCR反応時における増幅ミスの割合が低いKOD DNA Polymerase（東洋紡績株式会社製）等を用いることが好ましい。
【００４５】
PCRの反応条件は、上記染色体ＤＮＡ、プライマー、ＤＮＡポリメラーゼを用いることにより、tan遺伝子を含む特異的増幅産物を得ることができればいずれの反応条件を用いてもよい。その例としては、実施例１記載のPCR反応条件等が挙げられる。また、本発明のタンナーゼ遺伝子を有する微生物から染色体ＤＮＡを抽出する方法としては、公知のいずれの方法も使用できる。その例としては、飯村等の方法〔Agric. Biol. chem. 323-328, 51 (1987)〕等が挙げられる。
【００４６】
上記染色体ＤＮＡ、プライマー、ＤＮＡポリメラーゼを用いて、上記反応条件でPCRを実施することにより約1.8kb特異的増幅産物を得ることができる。該増幅産物の塩基配列は、染色体上の本発明のタンナーゼ遺伝子の塩基配列と同様の配列を有していることが期待される。しかしながら、場合によってはPCR反応中の増幅ミスにより、染色体上の本発明のタンナーゼ遺伝子の塩基配列と異なる塩基を有していることがあり、増幅ミスの有無を確認する必要がある。PCR反応中の増幅ミスの確認は、複数の増幅産物のシークエンスを行なうことにより以下の通り実施することができる。
【００４７】
個別のチューブを用いてPCRを行なうことにより得られた複数の増幅産物を、適当な制限酵素で処理・精製後、あるいは無処理で、ベクターＤＮＡ、例えば、プラスミドpUC18（タカラバイオ株式会社製）、pUC118（タカラバイオ株式会社製）、温度感受性バクテリオファージ（特開昭58-212781 号公報、FERM BP-133 等）の適当な制限酵素ゼ部位に挿入して組み換え体ＤＮＡを作成し、該組み換え体ＤＮＡを用いて、宿主細胞、例えば、大腸菌JM109（タカラバイオ株式会社製）を形質転換する。得られた形質転換体に含まれる組み換え体ＤＮＡを、QIAGEN Plasmid Mini Kit（フナコシ社製）等を用いて精製する。
【００４８】
このようにして得られた組み換え体ＤＮＡに挿入されている各増幅産物の塩基配列の決定は、ジデオキシ法(Methods in Enzymology,101,20-78,1983）等により行なうことができる。シークエンスを行なった各増幅産物の塩基配列のうち、全ての塩基について、対応する他の増幅産物の塩基と高い相同性を有している塩基配列を、染色体上の本発明のタンナーゼ遺伝子の塩基配列と同様の配列とみなすことができ、該塩基配列を有する増幅産物をtan遺伝子を含むＤＮＡとすることができる。該塩基配列を配列番号２に、配列番号２に示した塩基配列に基づいて確定した本発明の新規タンナーゼのアミノ酸配列を配列番号１に示す。
【００４９】
２．組み換え体ＤＮＡの構築と組み換え微生物の取得
本酵素を得るためには、tan遺伝子を含む組み換え体ＤＮＡを構築し、該組換え体ＤＮＡにより形質転換され、本酵素を生産し得る組み換え微生物を取得する必要がある。以下に、tan遺伝子を含む組み換え体ＤＮＡの構築と、該組換え体ＤＮＡを用いた微生物の形質転換方法について説明する。
【００５０】
形質転換に宿主として用いる微生物としては、tan遺伝子を含む組み換え体ＤＮＡによる形質転換により、本酵素を生産することができる微生物であればいかなる微生物も用いることができるが、好ましくは糸状菌、更に好ましくはアスペルギルス属に属する微生物、更に好ましくは黄麹菌、最も好ましくは、アスペルギルス・オリゼが挙げられる。
【００５１】
tan遺伝子を含む組み換え体ＤＮＡの構築に用いるベクターＤＮＡは、宿主細胞を形質転換する機能例えば、薬剤耐性、栄養要求性等のマーカー遺伝子、及び、宿主細胞で機能し得るプロモーター、ターミネーター等のtan遺伝子の発現に必要な機能を有している限りいかなるベクターＤＮＡも使用することができる。但し、tan遺伝子の発現に必要な機能については、挿入するtan遺伝子を含むＤＮＡ断片が、その機能を有している場合は必ずしも必要ではない。又、co-transformation法により形質転換を行なう場合には、ベクターＤＮＡ上に必ずしもマーカー遺伝子を有する必要はない。
【００５２】
tan遺伝子を含む組み換え体ＤＮＡは、PCR増幅ミスがないことを確認したtan遺伝子を含む増幅産物を上記ベクターＤＮＡとtan遺伝子の発現が可能な形で結合することにより構築することができる。すなわち、シークエンスに用いたtan遺伝子を含むプラスミドより、適当な制限酵素、例えば、EcoRＩ及びSmaＩで切り出したtan遺伝子を含むDNA断片を、実施例２記載のpAP等のベクターＤＮＡの適当な制限酵素部位、例えば、EcoRＩ-SmaＩ部位にtan遺伝子の発現が可能な形で挿入することにより構築することができるる。
【００５３】
tan遺伝子を含む組み換え体ＤＮＡを用いて微生物を形質転換する方法は、宿主として用いる微生物及びtan遺伝子を含む組み換え体ＤＮＡに用いたマーカー遺伝子により異なるが、公知の方法を適宜用いることができる。例えば、宿主としてアスペルギルス・オリゼniaD^-株を用い、マーカーとしてアスペルギルス・オリゼ由来のniaD遺伝子を用いる場合は、〔Mol.Gen.Genet (1989)218:99-104〕記載の方法を、宿主としてアスペルギルス・オリゼargB^-株を用い、マーカーとしてアスペルギルス・オリゼ由来のargB遺伝子を用いる場合は、〔Biosci.Biotechnol.Biochem.,64(4),816-827,2000〕記載の方法を用いることができる。
【００５４】
３．組み換え微生物による本酵素の生産
上記tan遺伝子を含む組み換え体ＤＮＡを保有する組み換え微生物を培養すれば、本酵素を生産することができる。培養方法は、通常の固体培養法でもよいが、液体培養法を採用することが好ましい。
【００５５】
組み換え微生物を培養する培地としては、例えば、酵母エキス、ペプトン、ソイトン、肉エキス、コーンスティープリカー又は大豆もしくは小麦ふすまの浸出液等から選ばれる１種以上の窒素源に、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、塩化第二鉄、硫酸第二鉄又は硫酸マンガン等の無機塩類の１種以上を添加し、更に必要により糖質原料、ビタミン、tan遺伝子を発現させるプロモーターを誘導するための基質等を適宜添加したものが用いられる。
【００５６】
培地の初発ｐＨは６〜７に調節するのが適当である。培養は、25〜35℃、好ましくは30℃前後で60〜100時間、通気撹拌培養、振盪培養、静置培養等により行なうことが好ましい。培養終了後、培養物より本酵素を採取するには、通常の酵素採取手段を用いることができる。すなわち、酵素が菌体外部に放出される場合には、濾過又は遠心分離等により菌体を分離して、酵素液を含む培養液を粗酵素液として回収する。また酵素が菌体内部に存在する場合には、超音波破砕機、フレンチプレス、リゾチーム等の細胞壁溶解酵素、界面活性剤等を用いて、濾過又は遠心分離等により分離した菌体から酵素を抽出し、次いで、濾過又は遠心分離等を用いて不溶物を除去することにより、本酵素を含有する粗酵素液を得ることができる。本発明では、このようにして得られた粗酵素液をそのままタンナーゼ標品としてもよく、以下の精製手法によりさらに純度を高めてもよい。
【００５７】
上記の粗酵素液から、本酵素を更に精製するには、通常のタンパク質精製法を使用することができる。具体的には、硫安塩析法、有機溶媒沈澱法、イオン交換クロマトグラフィー、疎水クロマトグラフィー、ゲルろ過クロマトグラフィー、吸着クロマトグラフィー、アフィニティークロマトグラフィー、電気泳動法等が、単独又は適宜組み合わせて用いられる。
【００５８】
本酵素は、広範な安定ｐＨ範囲並びに優れた熱安定性をともに兼ね備えている。従って、本酵素は、例えば、試薬、食品加工用助剤として利用する場合に、ｐＨ又は温度等の使用環境が変化したときの影響を受けにくく、安定性に優れている点で、公知のタンナーゼよりも優れている。また本酵素は、至適pH範囲が公知のものより中性域に存在しており、公知のタンナーゼでは充分な作用効果が期待できなかったpH域における新たな用途、利用できる食品のバリエーションを提供することが期待される。
【００５９】
例えば、弱酸性域でよく作用する公知のタンナーゼは、このpH領域に含まれる緑茶あるいは紅茶、また果汁等の飲料に対して使用する際には、反応至適pH付近で反応するため好適であった一方で、野菜汁や、抽出後に色調を強めるため中性付近までpH調整されたウーロン茶には、反応至適pHを外れていることから、活性が顕著に低下し、充分な効果を示せないか、必要な効果を得るためには多量の酵素を使用しなければならない、あるいは酵素処理のために一時的にpHを変える処理工程を必要とする等の問題を抱えていた。このようなpH領域の飲料・食品に対して本発明のタンナーゼを使用すれば、反応至適pH付近で反応するため、迅速に、より少量の酵素使用で、充分な効果をあげることが可能となる。
【００６０】
更に本酵素は、失活を起こす温度及び至適温度範囲が公知のタンナーゼより高温域に存在しており、食品加工等に使用するにあたり、加熱工程との共存がより容易であるという点でも、公知のタンナーゼでは行なえない使用法のバリエーションを提供する。例えば、茶飲料等は製法上、茶葉を熱湯抽出して製造するが、この茶抽出液にタンナーゼを作用させたい場合、公知のタンナーゼでは、酵素が熱失活しない温度まで充分に茶抽出液の温度を下げる必要があった。この点で、本発明の熱安定性に優れ、高温・中性域で良く反応するタンナーゼを使用する場合には、茶抽出液の温度を下げる工程を大幅に短縮するあるいは省くことが可能になる。
【００６１】
本発明のタンナーゼは、60℃〜80℃という高温で好適に作用するので、熱水抽出工程中に、抽出を行ないながら、酵素反応を行なわせることも可能になる。このことは、抽出及び酵素反応の２工程を1工程で済ませることにつながり、効率よい生産に寄与する。また、タンナーゼの作用の中に、茶抽出物中のタンニンを分解して沈澱や混濁を防止する効果があるが、抽出しながら酵素反応を進めることにより、沈澱・混濁を除去しつつ、より高濃度の茶抽出を実現することも可能になる。なお、本発明のタンナーゼは、実用上高い熱安定性を有しつつも、通常の飲料製造で広く用いられる加熱殺菌条件、例えば、達温95℃等の条件で、迅速に完全に失活するため、製造工程中、失活のために特別の加熱工程を組み入れる必要を生じない。
【００６２】
【実施例】
次に、本発明を実施例により更に具体的に説明する。但し、本発明の技術的範囲はこれら実施例に限定されるものではない。
【００６３】
〔実施例１〕
アスペルギルス・オリゼtan遺伝子の取得
アスペルギルス・オリゼ RIB40株(ATCC 42149)の胞子をYPD培地100 mlに植菌し、温度30℃で一晩振盪培養した。その後、飯村〔Argric. Biol. Chem. 323-328, 51 (1987)〕の方法に従ってゲノムＤＮＡを抽出し、このゲノムＤＮＡ断片を鋳型として、アスペルギルス・オリゼのゲノムデータベースを参考に作成した下記のプライマーを用いてPCRを行なった。
5'-cttggaattctctcgaataaaaatgagg-3' （配列番号 3）
5'-ctaaatacccgggctatgacatgacattc-3' （配列番号4 ）
PCR反応は、KOD DNA Polymerase (東洋紡績株式会社製)を使用し、PTC-200 DNA Engine(MJ Research社製)により行なった。反応液の組成は以下の通りである。
【００６４】
（試薬：使用量）
H₂O：36μl
10×Reaction Buffer #1：5μl
2 mMdNTP Mix：5μl
20μMプライマー2種類：各1μl
鋳型 (ＤＮＡ 0.5μg)：1μl
KOD DNA Polymerase：1μl
合計液量：50μl
【００６５】
上記の反応液50μlを0.2 ml反応チューブ中で混合してＤＮＡサーマルサイクラーにセットし、以下のような温度設定によりPCRを行なった。PCRにより得られた約1.8kbの増幅産物をエタノール沈殿処理後、EcoRI及びSmaIで消化した。
95℃、2分 ：1サイクル
95℃、30秒、 58℃、30秒 72℃、2分30秒 ：30サイクル
72℃、3分 ：1サイクル。
【００６６】
EcoRI及びSmaI消化後の上記増幅産物をEcoRI及びSmaIで消化後、精製したプラスミドpUC18のEcoRI-SmaI部位に連結し、大腸菌JM109(ATCC 53323)の形質転換及びクローニングを行なった。目的のＤＮＡ断片を有する大腸菌６クローンからプラスミドを調製し、クローニングしたＤＮＡ断片の塩基配列を解析した。各ＤＮＡ断片の塩基配列を比較し、PCR増幅時の増幅ミスを含まないと思われる増幅産物を含むプラスミドをpUCtan-4とした。pUCtan-4に挿入されたPCR増幅産物の塩基配列を配列番号２に示す。
【００６７】
〔実施例２〕
麹菌発現ベクターの構築
アスペルギルス・オリゼのアミラーゼプロモーターを有する発現ベクタープラスミドpMAR5(Biosci. Biotech. Biochem., 56:1674-1675, 1992)のマーカー遺伝子であるargB遺伝子を、アスペルギルス・オリゼのpyrG遺伝子と交換したプラスミドを作成した。pMAR5を制限酵素SphIで消化し、末端平滑化処理後、更にSalIで消化し、0.7%アガロースゲルで電気泳動し、約４kbの断片を回収した。
【００６８】
一方、ppyrG-26(特開2001-46053号公報記載)をBamHIで消化し、末端平滑化処理後、更に、SalIで消化し、0.7%アガロースゲルで電気泳動し、pyrG遺伝子を含む約2.2kbのＤＮＡ断片を回収した。これらの断片をライゲーションし、そのベクターにより大腸菌JM109株を形質転換した。得られたプラスミドをpAPとした。このプラスミドは、pyrG遺伝子を選択マーカーとして有し、アミラーゼ遺伝子のプロモーター及びターミネーターの間にEcoRI-SmaIサイトを有する発現ベクターであり、EcoRI-SmaIサイトにプロモーターと同じ方向で遺伝子のORFを組み込み、麹菌を形質転換することにより、アミラーゼ遺伝子プロモーターの制御下で目的の遺伝子を発現することができるものである。
【００６９】
pUCtan-4をEcoRI-SmaIで消化後、クローニングしたＤＮＡ断片を単離、精製し、pAPのEcoRI-SmaI部位に連結したのち大腸菌JM109の形質転換を行なった。得られた形質転換体より回収したプラスミドのうち、pAPのEcoRI-SmaI部位にクローニングしたＤＮＡ断片が目的通りに連結されたプラスミドをpAPtan-4とした。
【００７０】
〔実施例３〕
麹菌形質転換体の取得
アスペルギルス・オリゼ RIB40株(ATCC 42149)から特開2001-46053号公報記載方法で取得したpyrG欠損株をpAPtan-4で形質転換した。形質転換法は、プロトプラスト化した後ポリエチレングリコール及び塩化カルシウムを用いる方法（Mol. Gen. Genet., 218:99-104, 1989）によって行なった。pAPtan-4を3μg用いて形質転換し、最少培地で形質転換体を選択したところ、約100個のコロニーを得た。
【００７１】
2％デキストリン、1％ポリペプトン、0.5％ KH₂PO₄、0.05 ％MgSO₄を含むアミラーゼプロモーター誘導培地（pH6.0）50mlに、上記各形質転換体の分生子約10⁶個を接種し、温度30℃、50時間、振盪培養した。培養後の上清をUltrafree-MC（MILLIPORE社製）にかけ、菌体を完全に除去した後、各濾過液3μlをタンナーゼ活性アッセイプレート（0.2％ NH₄H₂PO₄、0.2％ KH₂PO₄、0.1％ MgSO₄、1％ Glucose、1％ Tannic acid、2％ agar、pH7.5）に滴下した。30℃で48時間静置培養した後、滴下した培養上清による培地中のタンニン酸の分解が観察された形質転換体RIB40tan-2株を選択した。
該形質転換株、アスペルギルス・オリゼ （RIB40tan-2）は、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-8180として寄託されている。
【００７２】
〔実施例４〕
組み換え微生物による本酵素の生産
(1) 種菌（胞子懸濁液）の調製
上記方法により作製した組み換え麹菌アスペルギルス・オリゼ（RIB40tan-2）を、マルツ寒天培地（純正化学社製）上で30℃、7日間培養した。得られたコロニー上に形成された胞子を、スキムミルク溶液（10％スキムミルク、5％サッカロース、1％グルタミン酸ナトリウム）で掻き取り、約10⁷個/mlの胞子懸濁液を調製して凍結保存した。
【００７３】
(2) 微生物の培養
酵素生産用培地（4％デキストリン、2％ポリペプトン、1％酵母エキス、0.5％ KH₂PO₄、0.05％MgSO₄、ｐＨ6.0）400mlを入れた2000ml容三角フラスコ5個に、アスペルギルス・オリゼ(RIB40tan-2)の胞子懸濁液0.8mlを植菌し、30℃で60時間振盪培養した後、濾紙濾過により菌体を除去し、培養液約1.8Ｌを得た。
【００７４】
(3)タンナーゼの精製
得られた培養液に硫安を添加し、飽和硫安濃度60％〜100％で回収される沈澱を緩衝液A［30％飽和硫安含有20mM 酢酸緩衝液（ｐＨ5.0）］にて溶解し、約130mlの酵素溶液を得た。次いで、該酵素溶液を、緩衝液Aで平衡化したフェニル-セファロースCL-4Bカラムに供した。カラムを緩衝液Aで洗浄した後、30%〜0％の飽和硫安含有20mM 酢酸緩衝液（ｐＨ5.0）の直線濃度勾配法により吸着画分を溶出した。溶出液を分取し、各画分のタンナーゼ活性を測定法１に従って調べた。タンナーゼ活性を有する画分を、セントリコン−30（アミコン社製）を用いて限外濃縮・透析し、硫安を除去した。
【００７５】
濃縮処理後の活性画分の一部を、0.1M NaCl含有20mM酢酸緩衝液pH5.5にて平衡化したTSK-gelG3000SWXL（東ソー社製）を用いたゲル濾過ＨＰＬＣに供し、溶出液を280nmの吸光度でモニターし、分取画分の活性を測定した。その結果、本酵素がシングルピークとなるまでに精製されていることを確認した。標準分子量マーカーの溶出位置から推定された本酵素の分子量は約25万であった。
【００７６】
また上記の方法により得られた活性画分の一部を、ＳＤＳ−ポリアクリルアミドゲル電気泳動に供した。泳動終了後にゲルの染色を行ない、本酵素が単一バンドとなるまでに精製されていることを確認した。本酵素のバンドはブロードになり、公知のタンナーゼ、例えば、アスペルギルス・オリゼ由来の酵素と同様に、糖鎖を有していることが確認された。分子量は約9〜10万であった。また、本酵素液の一部をN-グリコシダーゼ（グリコペプチダーゼＦ、タカラバイオ株式会社製）を用いて処理し、糖鎖を除去したものを同様にＳＤＳ−ポリアクリルアミドゲル電気泳動に供したものでは、ブロードなバンドは消失し、代わって分子量約6万のシャープな単一バンドが確認された。このことより、糖鎖のつかない本酵素蛋白質のサブユニット分子量は約6万であることが確認され、この分子量は、本発明中に開示した遺伝子の配列から算出される大きさと一致していた。
【００７７】
(4)タンナーゼの酵素化学的性質
本酵素と公知タンナーゼとの比較結果は、前記表１に示す通りである。表１から明らかなように、本酵素は、至適ｐＨ範囲がいずれの公知酵素よりも中性側にあり、中性条件下で良好に作用する。至適温度は公知酵素が中温域から上限60℃であるのに対し、60℃〜80℃と、顕著に高温域で良好に作用する。安定ｐＨ範囲は酸性域から弱アルカリ性に渡っており、広範囲である。そして、熱安定性についても、65℃で82％、70℃で63％、75℃で18％の活性を保持し、熱安定性に優れている。公知酵素はいずれも至適pHが弱酸性域にあり、本酵素が良好に作用する中性pH域での反応性は低い。
【００７８】
アスペルギルス・オリゼ由来の酵素（酵素Ａ）及びヨーロッパナラ由来の酵素（酵素Ｄ)の至適温度は40℃及び35℃〜40℃である。これらは中温域で良好に反応する一方、本酵素が良好に作用する高温域では反応性が顕著に低い。同時に、このような高温では活性が大きく低下するため、高温域では実用に供することができない。
【００７９】
アスペルギルス・アクレアタス由来の酵素（酵素Ｂ）及びバチルス・リケニフォルミス由来の酵素（酵素Ｃ）の至適温度は、やや高温域の50℃〜60℃にあり、アスペルギルス・オリゼ由来の酵素（酵素Ａ）あるいはヨーロッパナラ由来の酵素（酵素Ｄ)よりは高温域で使用することができる。しかし、茶飲料の熱抽出等に想定される、より高温域での使用は困難である。最も熱安定性の高いアスペルギルス・アクレアタス由来の酵素（酵素Ｂ）の細胞内酵素も、70℃で活性の70％を失うためである。また、その他の公知酵素同様に、至適pHは弱酸性域にあり、本酵素が良好に作用する中性pH域での反応性は低く、中性域で使用したい場合には、多量の酵素を使用しなければならない。更に重要なこととして、公知の全ての酵素は中性域での安定性に問題があるため、中性域で反応中に迅速に失活を起こし、目的の反応を完了することができなくなる恐れを有している。
【００８０】
上述のように本酵素は、至適pH及び至適温度範囲が公知の各種タンナーゼと異なっていることから、異なる用途、使用場面を提供し、特に公知酵素では効果を上げにくかった中性条件及び高温条件下の使用において特に優れていることは明らかである。更に、本酵素は、本酵素自身の至適pH及び至適温度範囲を外れた、例えば、公知酵素の至適pHである弱酸性条件及び公知酵素の至適温度範囲である中温域条件下においても、実用上充分な活性を有しているという点で、従来、公知酵素が用いられる場面においても、充分に使用可能である。
【００８１】
例えば、本酵素は60℃〜80℃でほぼ100％の活性を示すのに対し、40℃で50％、45℃で75％、50℃で80％の活性を示す。これは、本酵素が中温域から高温域まで広範な温度範囲で良好に反応できることを示す。また、本酵素はpH7.0〜7.5で100％の活性を示すのに対し、pH5.0でも38％、pH6.0では60％の活性を示す。これは、本酵素が弱酸性域においても実質的に良好に反応できることを示す。一方、公知のアスペルギルス・オリゼ由来の酵素（酵素Ａ）では60℃以上で酵素活性はなく、またpH7.0での活性は至適pH5.5の時の20％に過ぎない。このことから、本酵素は、その汎用性においても、公知酵素より優れている。即ち、これら各種の公知酵素に対し、本酵素は、各種食品加工の実際の使用工程中に利用するにあたり、優れている、また、公知酵素では好適に使用できなかった新たな使用場面を提供するものと考えられる。
【００８２】
以上より、本酵素は、試薬あるいは食品加工等の用途に利用する場合に、ｐＨ又は温度等の試験環境が変化したときの影響を受けにくく、その取扱いが容易で利用しやすいと考えられることから、公知のタンナーゼよりも優れているといえる。更に、汎用性が高く、公知のタンナーゼでは使用しにくかった新たな用途・使用工程に対しでも好適に使用することができ、タンナーゼの実用化のバリエーション化に貢献するものであるといえる。
【００８３】
【発明の効果】
本発明により、熱安定性に優れ、高温・中性域で良く反応するタンナーゼ及び該タンナーゼをコードする遺伝子が提供され、また、本酵素を効率よく大量に生産することができる。本酵素は熱安定性に優れ、中性域でよく作用し、また高温下でよく作用するという、新規な酵素化学的性質を有しているため、試薬、食品加工を始めとする各種産業分野において好適に使用できる。
【００８４】
【配列表】

【図面の簡単な説明】
【図１】本酵素の至適pH範囲を示す図。
【図２】本酵素の安定pH範囲を示す図。
【図３】本酵素の至適温度範囲を示す図。
【図４】本酵素の熱安定性範囲を示す図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a protein having tannase activity (hereinafter referred to as tannase), a gene thereof, and a method for producing tannase.
[0002]
[Prior art]
Tannase (tannin acylhydrolase, EC3.1.1.20) hydrolyzes tannins with depside bonds such as tannic acid, for example, m-di gallic acid (a kind of tannic acid in which two molecules of gallic acid are depside bonded) Catalyzes a reaction that hydrolyzes to produce two molecules of gallic acid. In the food industry, tannase is a useful enzyme used for anti-cream cream-down agent for tea or clarification during beer production.
[0003]
Conventionally, tannase activity has been reported from various natural microorganisms or plant leaves capable of degrading tannin. For example, Aspergillus genus [Aspergillus oryzae (see Non-patent document 1), Aspergillus niger (see Non-patent document 2), Aspergillus flavus (see Non-patent document 3), Aspergillus japonica (see Non-patent document 3)] Penicillium [Penicillium sp. (See Non-Patent Document 4)], Candida (see Non-Patent Document 5), Streptococcus bovis (see Non-Patent Document 6), Bacillus licheniformis (see Non-Patent Document 7), Lactobacillus Genus (see non-patent document 8), Klebsiella pneumoniae, corynebacterium sp. (See non-patent document 9), leaves of European oak (Quercus robur, syn. Q. pedunculate) (see non-patent document 10), etc. The derived tannase is known.
[0004]
Several tannases known in the art and whose properties have been clarified all have an optimum temperature range for reaction in the middle temperature range around 40 ° C, and the degree of thermal stability is lost at high temperatures exceeding 60 ° C. It is to live. Further, the optimum pH range for the reaction is a weakly acidic region in common with almost all, particularly around pH 5.0 to 5.5. Other tannases have been confirmed to be active, but there are no reports of tannase isolation and detailed enzymatic chemistry. That is, the conventional tannase has very few variations from the viewpoint of enzymatic chemistry.
[0005]
As a method for industrially mass-producing tannase, a method of culturing tannase-producing bacteria belonging to the genus Aspergillus in a medium and collecting tannase from the culture, for example, Patent Documents 1 and 2 are known. Enzymes produced by this method can be used in various applications, but naturally tannase produced has in common the enzymatic chemistry of tannase known in the art.
[0006]
[Patent Document 1]
Japanese Patent Publication No.56-8584
[Patent Document 2]
Japanese Patent Publication No. 7-79687
[0007]
[Non-Patent Document 1]
Agric. Biol. Chem. 36, 1553, 1972
[Non-Patent Document 2]
J. Biol. Chem. 14, 159, 1913
[Non-Patent Document 3]
Leather Sci., 24, 8, 1977
[Non-Patent Document 4]
Leather Chem., 11, 93, 1965
[Non-Patent Document 5]
Agric. Biol. Chem. 40, p.79-85
[Non-Patent Document 6]
Appl. Environ. Microbiol., 56, p.829-831
[Non-Patent Document 7]
J. Basic Microbiol., 40 (4), p.223-232, 2000
[Non-Patent Document 8]
Appl. Environ. Microbiol., Vol. 66, No. 7, p. 3093-3097, 2000
[Non-patent document 9]
J. Ferment. Technol., Vol. 61, No. 1, p. 55-59, 1983
[Non-Patent Document 10]
Phytochemistry. Vol. 45, No. 8, p. 1555-1560, 1997
[Non-Patent Document 11]
J. Basic Microbiol., 41 (6), p.313-318, 2001
[0008]
[Problems to be solved by the invention]
An object of the present invention is to provide tannase, its gene, and a method for producing tannase.
[0009]
[Means for Solving the Problems]
Accordingly, as a result of intensive studies, the present inventors have found a novel tannase that is significantly different from the conventionally reported tannases and succeeded in isolating the tannase gene. The present invention was completed by successfully culturing a microorganism containing a gene or a microorganism capable of producing tannase in a medium and collecting tannase from the obtained culture.
[0010]
That is, the present invention
1. A protein having tannase activity of (a) or (b) below,
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1
(b) a protein comprising an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence represented by SEQ ID NO: 1
2. A protein having an amino acid sequence having 65% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 1 and having tannase activity;
[0011]
3. A protein having the following physicochemical properties and tannase activity.
i) Action: Hydrolyzes tannin having a depside bond such as tannic acid.
ii) Optimum pH and stable pH range: The optimum pH is pH 7.0 to pH 7.5, and the stable pH range is pH 2.5 to pH 7.5 when treated at 30 ° C. for 80 minutes.
iii) Thermal stability: Residual activity after treatment for 10 minutes at 65 ° C. in citrate buffer (pH 5.5) is 80% or more.
iv) Optimal temperature range: 60 ° C to 80 ° C in citrate buffer (pH 5.5),
[0012]
4). A method for producing the protein, comprising culturing a microorganism having a protein-producing ability having tannase activity according to the above 1, 2, or 3 in a medium, and collecting tannase from the obtained culture;
5. A tannase gene encoding a protein having the tannase activity of (a) or (b) below,
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1
(b) a protein comprising an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence represented by SEQ ID NO: 1
[0013]
6). A tannase gene encoding a protein having an amino acid sequence of 65% or more of the amino acid sequence represented by SEQ ID NO: 1 and having tannase activity;
7). A tannase gene encoding a protein having tannase activity comprising the following DNA of (a) or (b):
(a) DNA consisting of the base sequence represented by SEQ ID NO: 2
(b) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 2 and encodes a protein having tannase activity
[0014]
8). Recombinant DNA in which the tannase gene according to 5, 6 or 7 is incorporated into vector DNA,
9. A method for producing the protein, which comprises culturing a microorganism containing Aspergillus genus and containing the recombinant DNA according to 8 above in a medium, and collecting a protein having tannase activity from the obtained culture;
It is.
In the present specification, the amino acid sequence represented by SEQ ID NO: 1 and the base sequence represented by SEQ ID NO: 2 are the sequences in the specification of the previous application 2001-403261 (filing date: December 27, 2001). The numbers 29158 and 29157 are the same.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The present invention identifies a gene having a base sequence different from a conventionally known tannase gene in a microorganism belonging to the genus Aspergillus used for fermentation, brewing, enzyme production and the like, and a recombinant vector containing the gene. By introducing it into microorganisms belonging to the genus Aspergillus, we succeeded in producing tannase with superior thermal stability and optimal temperature / pH in the high temperature / neutral range compared to the conventionally known tannase. Is. The tannase of the present invention is considered to be usable in a field where a conventionally known tannase cannot be used due to its enzymatic properties.
[0016]
[1] Novel tannase of the present invention
In a specific embodiment, the tannase of the present invention (hereinafter referred to as “the present enzyme” depending on the context) has the following physicochemical properties.
(1) Action: Hydrolyzes tannin having a depside bond such as tannic acid.
(Reaction example: Digallic acid + H ₂ O → 2 gallic acid)
(2) Optimal pH and stable pH range: The optimal pH of the present enzyme was determined by measuring the activity at each pH by changing the pH of the buffer used in measurement method 1 described later. The result is as shown in FIG. 1, and the optimum pH of the present enzyme is pH 6.5 to 8.0, particularly around pH 7.0 to 7.5. Further, the stable pH range was obtained by measuring the residual activity by measuring method 1 after treating the enzyme solution to be used in the range of pH 1.5 to 10.0 at 30 ° C. for 80 minutes. The result is as shown in FIG. 2, and the stable pH range of the present enzyme is pH 2.5 to 7.5.
[0017]
(3) Thermal stability: Residual activity after treatment for 10 minutes at each temperature in citrate buffer (pH 5.5) was examined according to measurement method 1. The results are as shown in FIG. 4. The enzyme is stable up to 60 ° C., beyond which it gradually begins to be deactivated, and is almost completely deactivated by heat treatment at 80 ° C. The residual activity at each temperature is 82% at 65 ° C, 63% at 70 ° C, and 18% at 75 ° C. (4) Optimal temperature range: The optimal temperature of the present enzyme was determined by measuring the activity at each temperature by changing the temperature during the reaction in Measurement Method 1 described later. The result is as shown in FIG. 3, and the optimum temperature of this enzyme is 60 ° C. to 80 ° C.
(5) Conditions for inactivation due to pH, temperature, etc .: This enzyme is stable at pH 2.5 to 7.5 as described above when treated with citrate buffer (pH 5.5) at 30 ° C. for 80 minutes. Deactivation is observed on the acidic side and alkaline side, and 20% or more is deactivated at pH 2.0 or lower and pH 8.0 or higher (activity measurement is according to measurement method 1).
[0018]
(6) Substrate specificity: acts on tannic acid, methyl gallate, ethyl gallate, propyl gallate.
It also acts on epigallocatechin gallate and epicatechin gallate. These substrates have been confirmed to work with known tannases such as Aspergillus oryzae-derived enzymes, and the action has been confirmed with this enzyme. Since there are so many kinds of tannins, the substrate specificity of tannins other than the above-mentioned substrates is not clear for known tannases as well, and this enzyme is also used for other tannins other than the above-mentioned substrates. It includes the possibility of acting.
(7) Molecular weight: As a result of measurement by gel filtration, the molecular weight is about 250,000, and as measured by SDS polyacrylamide gel electrophoresis, the molecular weight is 90,000 to 100,000, and it has a sugar chain.
[0019]
Table 1 shows differences in physicochemical properties between the present enzyme and conventionally known tannase. Enzyme A is derived from Aspergillus oryzae (described in the enzyme catalog of Kikkoman), enzyme B is derived from Aspergillus acreatas (see Non-Patent Document 11), and enzyme C is derived from Bacillus licheniformis (see Non-Patent Document 7). , And enzyme D mean tannase derived from European oak leaves (see Non-Patent Document 10).
[0020]
[Table 1]

[0021]
The tannase of the present invention is not limited by its origin as long as it has the physicochemical properties described herein, but is preferably a protein derived from a microorganism, more preferably a protein derived from a filamentous fungus, and more preferably Is a protein derived from a filamentous fungus belonging to the genus Aspergillus, and most preferably a protein derived from jaundice (Aspergillus oryzae, Aspergillus soja, Aspergillus tamari). Furthermore, the tannase of the present invention includes tannase obtained by gene recombination technology.
[0022]
In a specific embodiment, the protein having tannase activity of the present invention is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1. However, the protein having tannase activity of the present invention is not limited to the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 as long as it has tannase activity. That is, the protein having tannase activity of the present invention has one or more (preferably 2 to 50, more preferably 2 to 10, more preferably several) amino acids in the amino acid sequence represented by SEQ ID NO: 1. It may be a protein consisting of a deleted, substituted or added amino acid sequence and having tannase activity. Here, “the amino acid sequence in which one or more amino acids are added, deleted or substituted” means that the amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence as long as the desired tannase activity is obtained. Means that it may be added to a different sequence, and a part of the amino acid sequence may be deleted or substituted. For example, even if the first methionine of the amino acid sequence represented by SEQ ID NO: 1 is deleted, the tannase activity that is excellent in the thermal stability of the present invention and reacts well in the high temperature and neutral range can be obtained. Insofar as such deleted sequences are meant to be included in the present invention.
[0023]
Further, the protein having tannase activity of the present invention comprises a continuous amino acid sequence region occupying 30% or more of the entire amino acid sequence represented by SEQ ID NO: 1 and 65% or more, preferably 70% or more, more preferably 80% or more. It can be a protein consisting of an amino acid sequence showing sequence homology or a partial fragment thereof, and a protein having tannase activity. Furthermore, the protein having tannase activity of the present invention comprises a continuous amino acid sequence region occupying 60% or more of the entire amino acid sequence represented by SEQ ID NO: 1 and 65% or more, preferably 70% or more, more preferably 80% or more. Or a partial fragment thereof, and may be a protein having tannase activity.
[0024]
Furthermore, the protein having tannase activity of the present invention comprises a continuous amino acid sequence region occupying 90% or more of the entire amino acid sequence represented by SEQ ID NO: 1 and 65% or more, preferably 70% or more, more preferably 80% or more. It can be a protein consisting of an amino acid sequence showing sequence homology or a partial fragment thereof, and a protein having tannase activity.
Next, a method for measuring the titer of the enzyme will be described. The activity of this enzyme was measured according to the following measurement method 1. The amount of enzyme that hydrolyzes 1 μmol of ester bond in tannic acid at 30 ° C. per minute is defined as 1 unit (U).
[0025]
[Measurement Method 1] The method described in Agricultural Biological Chemistry (Vol. 31, 513-518, 1967) is partially modified to measure the titer of this enzyme. That is, the degree to which the ester bond of tannic acid (digallic acid) is hydrolyzed is calculated by measuring the decrease in absorbance at 310 nm spectrophotometrically.
[0026]
Specifically, 1.0 ml of the substrate solution [50 mM citrate buffer (pH 5.5) containing 0.35% tannic acid (digallic acid)] was equilibrated at 30 ° C. for 5 minutes and appropriately diluted tannase solution 0.25 ml And react at 30 ° C. for 15 minutes. Next, 5.0 ml of 90% ethanol is mixed to stop the reaction, 0.25 ml of this solution is taken and transferred to another test tube, and 5.0 ml of 90% ethanol is mixed and diluted. Next, the change in absorbance at 310 nm is measured using a spectrophotometer (U-2001, manufactured by Hitachi). A buffer solution is added instead of the enzyme and the same measurement is performed as a blank value, and the difference between the measured value and the blank value is defined as the enzyme reaction amount.
[0027]
[2] Novel tannase gene of the present invention
In a specific embodiment, the tannase gene of the present invention may be a tannase gene encoding a protein having the above physicochemical properties.
Furthermore, in a specific embodiment, the tannase gene of the present invention may be a tannase gene consisting of the base sequence represented by SEQ ID NO: 2. However, as long as it encodes a protein having tannase activity, it is not limited to the tannase gene consisting of the base sequence represented by SEQ ID NO: 2. That is, the tannase gene of the present invention hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 2 and encodes a protein having tannase activity. It can be a tannase gene.
[0028]
The stringent condition is a condition in which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal, and differs depending on the hybridization system to be used and the type, sequence and length of the probe. Such conditions can be determined by changing the hybridization temperature, washing temperature and salt concentration. For example, when a non-specific hybrid signal is strongly detected, the specificity can be increased by raising the hybridization and washing temperature and, if necessary, lowering the washing salt concentration. If no specific hybrid signal is detected, the hybrid can be stabilized by lowering the hybridization and washing temperatures and, if necessary, raising the washing salt concentration. Such optimization can be easily performed by researchers in this technical field.
[0029]
As specific examples of stringent conditions, for example, a DNA probe is used as a probe, and hybridization is performed using 5 × SSC, 1.0% (W / V) nucleic acid hybridization blocking reagent (Boehringer Mannheim), 0.1% (W / V) N-lauroyl sarcosine, 0.02% (W / V) SDS is used overnight (about 8 to 16 hours). Washing is performed twice for 15 minutes using 0.1 to 0.5 × SSC, 0.1% (W / V) SDS, preferably 0.1 × SSC, 0.1% (W / V) SDS. Hybridization and washing temperatures 65 ℃ or higher, preferably 68 ℃ or higher It is.
[0030]
In addition, the tannase gene of the present invention exhibits a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more with the DNA comprising the nucleotide sequence represented by SEQ ID NO: 2, and has the function of tannase. It may be a tannase gene that encodes a protein having the same.
[0031]
In the analysis of the amino acid sequence or base sequence described above, in order to determine the sequence homology between the two amino acid sequences or base sequences, the sequences are first pre-processed in an optimal state for comparison. For example, by making a gap in one sequence, the alignment with the other sequence is optimized. Thereafter, the amino acid residues or bases at each site are compared. When the same amino acid residue or base is present at a site in the first sequence as the corresponding site in the second sequence, the sequences are identical at that site.
Sequence homology between the two sequences is expressed as a percentage of the total number of sites (all amino acids or all bases) of the number of sites that are identical between the sequences.
[0032]
In accordance with the above principle, sequence homology between two amino acid sequences or base sequences is determined by the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990 and Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). A BLAST program using such an algorithm was developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Furthermore, Gapped BLAST is a program for determining sequence homology more sensitively than BLAST (Nucleic Acids Res. 25: 3389-3402, 1997). The above program is mainly used to search a database for sequences showing high sequence homology to a given sequence. These are available, for example, on the Internet website of the National Center for Biotechnology Information.
[0033]
However, if a sequence showing significant sequence homology is not found in the BLAST software, a more sensitive FASTA software (WR Pearson and DJ Lipman, Proc. Natl. Acad. Sci., 85: 2444-2448, 1988 ) Can be used to search the database for sequences showing sequence homology. The FASTA software can be used, for example, on the GenomeNet website. Again, default parameters are used. For example, when searching for a base sequence, nr-nt is used in the database and a ktup value of 6 is used.
[0034]
Each of the above methods is mainly used for searching a sequence showing sequence homology from a database. In the present invention, Genetyx Mac Ver (Toware Development Co., Ltd.) is used as a means for determining sequence homology of individual sequences. ) Homology analysis. This method is based on the Lipman-Pearson method (Science, 227: 1435-1441, 1985), which is frequently used as a high-speed and high-sensitivity method. When analyzing the sequence homology of a base sequence, a region encoding a protein (CDS or ORF) is used if possible. As parameters, Unit Size to compare = 2, Pickup Location = 5, and the result is displayed in%. If the sequence homology of the alignment that showed the highest point is used as a result, but does not show more than 30%, 50%, or 70% overlap of the query sequence, it is functionally correlated Since it is not necessarily estimated, it is not used as a value indicating sequence homology between two sequences. For example, even if there is a perfect match region of about several bases / residues, it is highly likely that this is only a coincidence result, and the match in the length of about several percent of the whole includes a specific functional motif. Even if it is, it is difficult to think that it performs the same function as a whole.
[0035]
[3] Production of this enzyme using microorganisms
Any microorganism may be used as a microorganism used for producing the present enzyme as long as it has the ability to produce the enzyme, preferably with or without genetic recombination, preferably a filamentous fungus, more preferably Aspergillus. Filamentous fungi belonging to the genus are used. Specifically, Aspergillus oryzae RIB40 (ATCC 42149) etc. can be mentioned, for example. It is also possible to use a mutant strain of the strain. As a method for obtaining a mutant strain of the strain, for example, a method of causing mutation in the original strain using a radioisotope, ultraviolet light, nitrosoguanidine or the like can be used.
[0036]
As a matter of course, the microorganism used for the production of the present enzyme may have the ability to produce the present enzyme and at the same time have, for example, the ability to produce conventional tannase. Such microorganisms can be used to produce multiple types of tannase by introducing a gene that encodes another type of tannase into one that originally has both tannase-producing ability, or a microorganism that originally has the ability to produce a certain type of tannase. The ones that have the ability are included. By culturing a microorganism having the ability to produce multiple types of tannase under various culture conditions, it is possible to produce enzymes by arbitrarily changing the production ratio of each tannase according to the purpose.
[0037]
The above microorganism is cultured in a medium, and the enzyme is collected from the resulting culture. The culture method may be a normal solid culture method, and preferably employs a liquid culture method. As the medium, any of a synthetic medium or a natural medium can be used as long as it contains a normal medium for culturing microorganisms, that is, a carbon source, a nitrogen source, an inorganic substance, and other nutrients in an appropriate ratio.
[0038]
As the carbon source, assimilated carbon compounds such as glucose, maltose, starch hydrolysate, glycerin, fructose, molasses and the like are used. As the nitrogen source, usable nitrogen compounds such as yeast extract, peptone, soyton, meat extract, corn steep liquor, soybean flour, soybean or wheat bran leachate, amino acid, ammonium sulfate, ammonium nitrate and the like are used. . In addition, various salts such as salt, potassium chloride, magnesium sulfate, manganese chloride, ferrous sulfate, ferrous potassium phosphate, dipotassium phosphate, sodium carbonate, sodium acetate, vitamins, antifoaming agents, etc. are used. Is done. These medium components can be used alone or in appropriate combination.
[0039]
The culture of microorganisms is appropriately selected from aeration and agitation culture, shaking culture, stationary culture and the like according to the properties of the microorganisms used and the shape of the medium, and the initial pH of the medium is adjusted to about 6 to 7, followed by 25 to 35. C., preferably around 30.degree. C., for 24 to 140 hours, preferably 60 to 100 hours. In order to collect the present enzyme from the culture after completion of the culture, usual enzyme collecting means can be used. That is, when the enzyme is released to the outside of the microbial cell, the microbial cell can be separated by filtration or centrifugation, and the culture solution containing the enzyme solution can be collected. In addition, when the enzyme is present inside the cell, the enzyme is extracted from the cell separated by filtration or centrifugation using a cell wall lytic enzyme such as an ultrasonic crusher, French press, lysozyme, or a surfactant. Then, a crude enzyme solution containing the present enzyme can be obtained by removing insolubles using filtration or centrifugation.
[0040]
In order to further purify the present enzyme from the crude enzyme solution thus obtained, an ordinary protein purification method can be used. Specifically, for example, ammonium sulfate salting-out method, organic solvent precipitation method, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, adsorption chromatography, affinity chromatography, electrophoresis method, etc. alone or in appropriate combination Can be used.
[0041]
[4] Cloning of the enzyme gene and production of the enzyme by genetic engineering techniques
In the following, a host cell is transformed or transduced with a recombinant DNA into which a DNA containing a gene encoding a book (hereinafter referred to as “tan gene” depending on the context) is inserted. The method of producing the will be described in detail.
[0042]
1. Cloning of tan gene
DNA containing the tan gene can be obtained by cloning a chromosomal DNA or a wild-type gene derived from cDNA. Alternatively, the gene can be amplified by performing a polymerase chain reaction (hereinafter referred to as “PCR”) using chromosomal DNA or cDNA as a template. Furthermore, it is possible to construct the gene using a chemical synthesis method. Hereinafter, a method for cloning a tan gene by PCR using chromosomal DNA of a microorganism belonging to the genus Aspergillus as a template will be described as an example of a method for isolating DNA containing the tan gene.
[0043]
The chromosomal DNA used as the template DNA for the amplification of the tan gene by the PCR method can be any chromosomal DNA derived from a microorganism as long as it is derived from a microorganism having the tannase gene of the present invention. Examples of these microorganisms include Aspergillus oryzae RIB40 (ATCC 42149) strain. As the primer, a primer having any sequence may be used as long as a DNA fragment containing the tan gene can be amplified. Examples thereof include primers represented by SEQ ID NOs: 3 and 4.
[0044]
For example, TaKaRa Ex Taq (manufactured by Takara Bio Inc.), KOD DNA Polymerase (manufactured by Toyobo Co., Ltd.) or the like can be used as the DNA polymerase used in the PCR reaction. It is preferable to use KOD DNA Polymerase (manufactured by Toyobo Co., Ltd.) and the like having a low viscosity.
[0045]
Any PCR reaction conditions may be used as long as a specific amplification product containing the tan gene can be obtained by using the chromosomal DNA, primer, and DNA polymerase. Examples thereof include the PCR reaction conditions described in Example 1. As a method for extracting chromosomal DNA from the microorganism having the tannase gene of the present invention, any known method can be used. Examples thereof include the method of Iimura et al. [Agric. Biol. Chem. 323-328, 51 (1987)].
[0046]
An amplification product specific for about 1.8 kb can be obtained by performing PCR under the above reaction conditions using the above chromosomal DNA, primer, and DNA polymerase. The base sequence of the amplified product is expected to have the same sequence as the base sequence of the tannase gene of the present invention on the chromosome. However, in some cases, due to an amplification mistake during the PCR reaction, there may be a base different from the base sequence of the tannase gene of the present invention on the chromosome, and it is necessary to confirm the presence or absence of the amplification mistake. Confirmation of an amplification error during a PCR reaction can be performed as follows by sequencing a plurality of amplification products.
[0047]
A plurality of amplification products obtained by performing PCR using individual tubes, after treatment and purification with appropriate restriction enzymes, or without treatment, vector DNA, for example, plasmid pUC18 (manufactured by Takara Bio Inc.), pUC118 (manufactured by Takara Bio Inc.), temperature-sensitive bacteriophage (JP 58-212781 A, FERM BP-133, etc.) is inserted into an appropriate restriction enzyme site to prepare a recombinant DNA, and the recombinant The host cell, for example, E. coli JM109 (manufactured by Takara Bio Inc.) is transformed with the DNA. Recombinant DNA contained in the obtained transformant is purified using QIAGEN Plasmid Mini Kit (manufactured by Funakoshi) or the like.
[0048]
The base sequence of each amplification product inserted in the recombinant DNA thus obtained can be determined by the dideoxy method (Methods in Enzymology, 101, 20-78, 1983) or the like. Among the base sequences of each amplification product subjected to sequencing, the base sequence having high homology with the bases of other corresponding amplification products is the base sequence of the tannase gene of the present invention on the chromosome. The amplification product having the base sequence can be a DNA containing the tan gene. The base sequence is shown in SEQ ID NO: 2, and the amino acid sequence of the novel tannase of the present invention determined based on the base sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1.
[0049]
2. Construction of recombinant DNA and acquisition of recombinant microorganisms
In order to obtain this enzyme, it is necessary to construct a recombinant DNA containing the tan gene and obtain a recombinant microorganism which can be transformed with the recombinant DNA and produce this enzyme. Below, the construction of recombinant DNA containing the tan gene and a method for transforming microorganisms using the recombinant DNA will be described.
[0050]
As a microorganism used as a host for transformation, any microorganism can be used as long as it can produce this enzyme by transformation with a recombinant DNA containing a tan gene, preferably a filamentous fungus, more preferably Is a microorganism belonging to the genus Aspergillus, more preferably japonicum, and most preferably Aspergillus oryzae.
[0051]
The vector DNA used for the construction of recombinant DNA containing the tan gene has functions for transforming host cells, for example, marker genes such as drug resistance and auxotrophy, and tan genes such as promoters and terminators that can function in host cells. Any vector DNA can be used as long as it has a function necessary for the expression of. However, the function necessary for expression of the tan gene is not necessarily required when the DNA fragment containing the tan gene to be inserted has the function. In addition, when transformation is performed by the co-transformation method, it is not always necessary to have a marker gene on the vector DNA.
[0052]
Recombinant DNA containing the tan gene can be constructed by combining an amplification product containing the tan gene, which has been confirmed to have no PCR amplification errors, in a form that allows expression of the tan gene. That is, an appropriate restriction enzyme site of a vector DNA such as pAP described in Example 2 is obtained by using a DNA fragment containing a tan gene excised with an appropriate restriction enzyme, for example, EcoRI and SmaI, from a plasmid containing a tan gene used in the sequence. For example, it can be constructed by inserting into the EcoRI-SmaI site in a form that allows expression of the tan gene.
[0053]
A method for transforming a microorganism using a recombinant DNA containing a tan gene varies depending on the microorganism used as a host and the marker gene used for the recombinant DNA containing a tan gene, but a known method can be used as appropriate. For example, Aspergillus oryzae niaD as a host ^- When using a niaD gene derived from Aspergillus oryzae as a marker, the method described in [Mol.Gen.Genet (1989) 218: 99-104] is used as the host, and Aspergillus oryzae argB is used as a host. ^- When the strain is used and the argB gene derived from Aspergillus oryzae is used as a marker, the method described in [Biosci. Biotechnol. Biochem., 64 (4), 816-827, 2000] can be used.
[0054]
3. Production of this enzyme by recombinant microorganisms
This enzyme can be produced by culturing a recombinant microorganism having a recombinant DNA containing the tan gene. The culture method may be a normal solid culture method, but a liquid culture method is preferably employed.
[0055]
As a medium for culturing the recombinant microorganism, for example, one or more nitrogen sources selected from yeast extract, peptone, soyton, meat extract, corn steep liquor, soybean or wheat bran leachate, potassium dihydrogen phosphate, phosphorus Add one or more inorganic salts such as dipotassium oxyhydrogen, magnesium sulfate, ferric chloride, ferric sulfate, or manganese sulfate, and if necessary, induce a promoter that expresses sugar raw materials, vitamins, and tan genes For this purpose, an appropriate addition of a substrate or the like is used.
[0056]
It is appropriate to adjust the initial pH of the medium to 6-7. The culture is preferably performed at 25 to 35 ° C., preferably around 30 ° C., for 60 to 100 hours by aeration and agitation culture, shaking culture, stationary culture, or the like. In order to collect the enzyme from the culture after completion of the culture, usual enzyme collecting means can be used. That is, when the enzyme is released to the outside of the microbial cell, the microbial cell is separated by filtration or centrifugation, and the culture solution containing the enzyme solution is recovered as a crude enzyme solution. In addition, when the enzyme is present inside the cell, the enzyme is extracted from the cell separated by filtration or centrifugation using a cell wall lytic enzyme such as an ultrasonic crusher, French press, lysozyme, or a surfactant. Then, a crude enzyme solution containing the present enzyme can be obtained by removing insolubles using filtration or centrifugation. In the present invention, the crude enzyme solution thus obtained may be used as a tannase preparation as it is, and the purity may be further increased by the following purification method.
[0057]
In order to further purify the present enzyme from the above crude enzyme solution, an ordinary protein purification method can be used. Specifically, ammonium sulfate salting-out method, organic solvent precipitation method, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, adsorption chromatography, affinity chromatography, electrophoresis method, etc. are used alone or in appropriate combination. .
[0058]
This enzyme has both a wide stable pH range and excellent thermal stability. Therefore, the present enzyme is a known tannase in that it is not easily affected by changes in the use environment such as pH or temperature when it is used as a reagent or a food processing aid, and is excellent in stability. Better than. In addition, this enzyme has an optimum pH range in a neutral range than known ones, and provides a new use in pH ranges where the known tannase could not be expected to have a sufficient effect, and variations in foods that can be used. Is expected to do.
[0059]
For example, a known tannase that works well in a weakly acidic region is suitable because it reacts near the optimum pH when used for beverages such as green tea, black tea, and fruit juice contained in this pH region. On the other hand, vegetable juice and oolong tea whose pH has been adjusted to near neutrality to enhance the color tone after extraction are not optimal for reaction because the pH is not optimal for the reaction. However, in order to obtain a necessary effect, a large amount of enzyme must be used, or a treatment step for temporarily changing the pH is required for the enzyme treatment. If the tannase of the present invention is used for beverages and foods in such a pH range, the reaction will occur near the optimum pH of the reaction, so that a sufficient effect can be obtained quickly and with a smaller amount of enzyme. Become.
[0060]
Furthermore, the present enzyme has an inactivation temperature and an optimum temperature range in a higher temperature range than known tannase, and it is easier to coexist with a heating step when used for food processing, etc. It provides variations in usage that are not possible with known tannases. For example, tea beverages and the like are manufactured by extracting tea leaves with hot water in the manufacturing method. When tannase is allowed to act on this tea extract, a known tannase is sufficient for the tea extract to a temperature at which the enzyme is not heat-inactivated. It was necessary to lower the temperature. In this regard, when using tannase which is excellent in heat stability of the present invention and reacts well in high temperature and neutral range, the process of lowering the temperature of the tea extract can be greatly shortened or omitted. .
[0061]
Since the tannase of the present invention suitably acts at a high temperature of 60 ° C. to 80 ° C., the enzymatic reaction can be performed while performing extraction during the hot water extraction step. This leads to two steps of extraction and enzyme reaction in one step, and contributes to efficient production. In addition, the action of tannase has the effect of decomposing tannin in tea extract to prevent precipitation and turbidity. It is also possible to achieve concentration tea extraction. The tannase of the present invention is rapidly and completely deactivated under heat sterilization conditions widely used in normal beverage production, for example, a temperature of 95 ° C. or the like, while having practically high heat stability. Therefore, it is not necessary to incorporate a special heating process for deactivation during the manufacturing process.
[0062]
【Example】
Next, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[0063]
[Example 1]
Acquisition of Aspergillus oryzae tan gene
Spores of Aspergillus oryzae RIB40 strain (ATCC 42149) were inoculated into 100 ml of YPD medium and cultured with shaking at a temperature of 30 ° C. overnight. Subsequently, genomic DNA was extracted according to the method of Iimura [Argric. Biol. Chem. 323-328, 51 (1987)], and the following primers were created using this genomic DNA fragment as a template with reference to the genome database of Aspergillus oryzae. PCR was performed using.
5'-cttggaattctctcgaataaaaatgagg-3 '(SEQ ID NO: 3)
5'-ctaaatacccgggctatgacatgacattc-3 '(SEQ ID NO: 4)
PCR reaction was performed using POD-200 DNA Engine (manufactured by MJ Research) using KOD DNA Polymerase (manufactured by Toyobo Co., Ltd.). The composition of the reaction solution is as follows.
[0064]
(Reagent: Amount used)
H ₂ O: 36 μl
10 x Reaction Buffer # 1: 5 μl
2 mM dNTP Mix: 5 μl
2 types of 20μM primers: 1μl each
Template (DNA 0.5μg): 1μl
KOD DNA Polymerase: 1μl
Total liquid volume: 50μl
[0065]
50 μl of the above reaction solution was mixed in a 0.2 ml reaction tube and set in a DNA thermal cycler, and PCR was performed with the following temperature settings. An amplification product of about 1.8 kb obtained by PCR was subjected to ethanol precipitation and then digested with EcoRI and SmaI.
95 ° C, 2 minutes: 1 cycle
95 ° C, 30 seconds, 58 ° C, 30 seconds 72 ° C, 2 minutes 30 seconds: 30 cycles
72 ° C, 3 minutes: 1 cycle.
[0066]
The amplified product after digestion with EcoRI and SmaI was digested with EcoRI and SmaI, then ligated to the EcoRI-SmaI site of the purified plasmid pUC18, and E. coli JM109 (ATCC 53323) was transformed and cloned. A plasmid was prepared from 6 E. coli clones having the target DNA fragment, and the base sequence of the cloned DNA fragment was analyzed. The nucleotide sequences of the DNA fragments were compared, and a plasmid containing an amplification product that was considered not to contain an amplification error during PCR amplification was designated as pUCtan-4. The base sequence of the PCR amplification product inserted into pUCtan-4 is shown in SEQ ID NO: 2.
[0067]
[Example 2]
Construction of koji mold expression vector
A plasmid was prepared by replacing the argB gene, which is a marker gene of the expression vector plasmid pMAR5 (Biosci. Biotech. Biochem., 56: 1674-1675, 1992) having the amylase promoter of Aspergillus oryzae, with the pyrG gene of Aspergillus oryzae. . pMAR5 was digested with the restriction enzyme SphI, blunt-ended, and further digested with SalI, and electrophoresed on a 0.7% agarose gel to recover a fragment of about 4 kb.
[0068]
On the other hand, pyrrG-26 (described in JP-A-2001-46053) is digested with BamHI, and after end blunting, further digested with SalI, electrophoresed on a 0.7% agarose gel, and containing about 2.2 kb containing the pyrG gene. DNA fragments were recovered. These fragments were ligated, and Escherichia coli JM109 strain was transformed with the vector. The obtained plasmid was designated as pAP. This plasmid is an expression vector having the pyrG gene as a selection marker and having an EcoRI-SmaI site between the promoter and terminator of the amylase gene, incorporating the ORF of the gene in the same direction as the promoter in the EcoRI-SmaI site, Is capable of expressing the target gene under the control of the amylase gene promoter.
[0069]
After digesting pUCtan-4 with EcoRI-SmaI, the cloned DNA fragment was isolated and purified, ligated to the EcoRI-SmaI site of pAP, and then transformed into E. coli JM109. Among the plasmids recovered from the obtained transformants, a plasmid in which the DNA fragment cloned into the EcoRI-SmaI site of pAP was ligated as intended was designated as pAPtan-4.
[0070]
Example 3
Acquisition of gonococcal transformants
A pyrG deficient strain obtained from Aspergillus oryzae RIB40 strain (ATCC 42149) by the method described in JP-A-2001-46053 was transformed with pAPtan-4. The transformation method was performed by a method using polyethylene glycol and calcium chloride after protoplastization (Mol. Gen. Genet., 218: 99-104, 1989). When transformation was performed using 3 μg of pAPtan-4, and transformants were selected with a minimal medium, about 100 colonies were obtained.
[0071]
2% dextrin, 1% polypeptone, 0.5% KH ₂ PO _Four 0.05% MgSO _Four About 10 conidia of each of the above transformants in 50 ml of amylase promoter induction medium (pH 6.0) containing ⁶ The seeds were inoculated and cultured with shaking at 30 ° C. for 50 hours. After culturing, the supernatant was applied to Ultrafree-MC (MILLIPORE) to completely remove the cells, and 3 μl of each filtrate was added to tannase activity assay plate (0.2% NH). _Four H ₂ PO _Four , 0.2% KH ₂ PO _Four , 0.1% MgSO _Four 1% Glucose, 1% Tannic acid, 2% agar, pH 7.5). After stationary culture at 30 ° C. for 48 hours, a transformant RIB40tan-2 strain in which degradation of tannic acid in the medium was observed by the dropped culture supernatant was selected.
The transformed strain, Aspergillus oryzae (RIB40tan-2) has been deposited as FERM BP-8180 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
[0072]
Example 4
Production of this enzyme by recombinant microorganisms
(1) Preparation of inoculum (spore suspension)
The recombinant Aspergillus oryzae (RIB40tan-2) produced by the above method was cultured on Marz agar medium (manufactured by Junsei Kagaku) at 30 ° C. for 7 days. Spores formed on the obtained colonies were scraped with a skim milk solution (10% skim milk, 5% sucrose, 1% sodium glutamate), and about 10 ⁷ Individual / ml spore suspension was prepared and stored frozen.
[0073]
(2) Microbial culture
Medium for enzyme production (4% dextrin, 2% polypeptone, 1% yeast extract, 0.5% KH ₂ PO _Four 0.05% MgSO _Four , PH 6.0) Inoculate 0.8 ml of Aspergillus oryzae (RIB40tan-2) spore suspension into five 2000 ml Erlenmeyer flasks containing 400 ml, shake culture at 30 ° C for 60 hours, and filter by filter paper The cells were removed to obtain about 1.8 L of culture solution.
[0074]
(3) Purification of tannase
Ammonium sulfate is added to the obtained culture broth, and the precipitate recovered at a saturated ammonium sulfate concentration of 60% to 100% is dissolved in buffer A [20% acetic acid buffer solution (pH 5.0) containing 30% saturated ammonium sulfate]. 130 ml enzyme solution was obtained. The enzyme solution was then applied to a phenyl-Sepharose CL-4B column equilibrated with buffer A. After the column was washed with buffer A, the adsorbed fraction was eluted by a linear concentration gradient method using 20 mM acetic acid buffer (pH 5.0) containing 30% to 0% saturated ammonium sulfate. The eluate was collected and the tannase activity of each fraction was examined according to measurement method 1. The fraction having tannase activity was subjected to ultraconcentration and dialysis using Centricon-30 (Amicon) to remove ammonium sulfate.
[0075]
A part of the active fraction after concentration treatment was subjected to gel filtration HPLC using TSK-gel G3000SWXL (manufactured by Tosoh Corporation) equilibrated with 20 mM acetate buffer pH 5.5 containing 0.1 M NaCl, and the eluate was 280 nm. The activity of the fractionated fraction was measured by monitoring the absorbance. As a result, it was confirmed that the enzyme was purified to a single peak. The molecular weight of the enzyme estimated from the elution position of the standard molecular weight marker was about 250,000.
[0076]
A part of the active fraction obtained by the above method was subjected to SDS-polyacrylamide gel electrophoresis. After the electrophoresis was completed, the gel was stained to confirm that the enzyme had been purified to a single band. The band of this enzyme was broad, and it was confirmed that the enzyme had a sugar chain as in the case of a known tannase such as an enzyme derived from Aspergillus oryzae. The molecular weight was about 90-100,000. In addition, a part of this enzyme solution treated with N-glycosidase (Glycopeptidase F, manufactured by Takara Bio Inc.) and the sugar chain removed is similarly subjected to SDS-polyacrylamide gel electrophoresis. The broad band disappeared, and a sharp single band with a molecular weight of about 60,000 was confirmed instead. From this, it was confirmed that the subunit molecular weight of the enzyme protein without a sugar chain was about 60,000, and this molecular weight was consistent with the size calculated from the gene sequence disclosed in the present invention. .
[0077]
(4) Enzymatic properties of tannase
The comparison results between this enzyme and known tannase are as shown in Table 1 above. As is clear from Table 1, the present enzyme has an optimum pH range on the neutral side with respect to any known enzyme and works well under neutral conditions. The optimum temperature is 60 ° C. to 80 ° C., while the known enzyme has an upper limit of 60 ° C. from the middle temperature range, and works well in the high temperature range. The stable pH range extends from acidic to weakly alkaline and is a wide range. As for thermal stability, the activity of 82% at 65 ° C, 63% at 70 ° C, and 18% at 75 ° C is maintained, and the thermal stability is excellent. All known enzymes have an optimum pH in a weakly acidic range, and have low reactivity in a neutral pH range where this enzyme works well.
[0078]
The optimum temperatures of the enzyme derived from Aspergillus oryzae (enzyme A) and the enzyme derived from European oak (enzyme D) are 40 ° C. and 35 ° C. to 40 ° C. While these react well in the middle temperature range, the reactivity is remarkably low in the high temperature range where the present enzyme works well. At the same time, the activity is greatly reduced at such a high temperature and cannot be put to practical use in a high temperature region.
[0079]
The optimal temperature of the enzyme derived from Aspergillus acreata (enzyme B) and the enzyme derived from Bacillus licheniformis (enzyme C) is 50 ° C to 60 ° C in a slightly high temperature range, and the enzyme derived from Aspergillus oryzae (enzyme A) or It can be used in a higher temperature range than the European oak-derived enzyme (enzyme D). However, it is difficult to use in a higher temperature range assumed for heat extraction of tea beverages. This is because the intracellular enzyme of the enzyme (enzyme B) derived from Aspergillus acreata having the highest thermostability also loses 70% of the activity at 70 ° C. In addition, like other known enzymes, the optimum pH is in the weakly acidic range, the reactivity in the neutral pH range where this enzyme works well is low, and if you want to use it in the neutral range, Must be used. More importantly, all known enzymes have problems with stability in the neutral range, which may cause rapid deactivation during the reaction in the neutral range and fail to complete the desired reaction. have.
[0080]
As described above, the present enzyme is different in the optimum pH and temperature range from various known tannases, and thus provides different uses and usage scenes. It is clear that it is particularly excellent in use under high temperature conditions. Furthermore, the present enzyme is outside the optimum pH and temperature range of the enzyme itself, for example, under weakly acidic conditions that are the optimum pH of the known enzyme and medium temperature range conditions that are the optimum temperature range of the known enzyme. However, since it has a practically sufficient activity, it can be sufficiently used even in a situation where a known enzyme is conventionally used.
[0081]
For example, the enzyme exhibits almost 100% activity at 60 ° C. to 80 ° C., whereas it exhibits 50% activity at 40 ° C., 75% at 45 ° C., and 80% activity at 50 ° C. This shows that this enzyme can react well in a wide temperature range from a medium temperature range to a high temperature range. In addition, this enzyme exhibits 100% activity at pH 7.0 to 7.5, whereas it exhibits 38% activity at pH 5.0 and 60% activity at pH 6.0. This indicates that the enzyme can react substantially well even in a weakly acidic region. On the other hand, the known enzyme derived from Aspergillus oryzae (enzyme A) has no enzyme activity at 60 ° C. or higher, and the activity at pH 7.0 is only 20% at the optimum pH 5.5. From this, this enzyme is superior to a known enzyme also in its versatility. That is, in contrast to these various known enzymes, the present enzyme is excellent in use during the actual use process of various food processing, and provides a new use scene that could not be suitably used with known enzymes. It is considered a thing.
[0082]
From the above, it is considered that this enzyme is not easily affected by changes in the test environment such as pH or temperature when used for applications such as reagents or food processing, and is easy to handle and use. It can be said that it is superior to known tannase. Furthermore, it is highly versatile and can be suitably used for new applications and processes that are difficult to use with known tannase, and it can be said that it contributes to variations in practical use of tannase.
[0083]
【The invention's effect】
According to the present invention, a tannase excellent in thermal stability and reacts well in a high temperature / neutral region and a gene encoding the tannase are provided, and the enzyme can be efficiently produced in large quantities. Since this enzyme has a novel enzymatic chemistry that is excellent in heat stability, works well in the neutral range, and works well at high temperatures, it can be used in various industrial fields including reagents and food processing. Can be preferably used.
[0084]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a graph showing the optimum pH range of the enzyme.
FIG. 2 is a view showing a stable pH range of the present enzyme.
FIG. 3 is a graph showing the optimum temperature range of the present enzyme.
FIG. 4 is a graph showing the thermostability range of the present enzyme.

Claims (5)

A protein having the following tannase activity (a) or (b):
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1
(b) a protein comprising an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence represented by SEQ ID NO: 1 A tannase gene encoding a protein having the following tannase activity (a) or (b):
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1
(b) a protein comprising an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence represented by SEQ ID NO: 1 The tannase gene which codes the protein which has the tannase activity which consists of DNA of the following (a) or (b).
(a) DNA comprising the base sequence represented by SEQ ID NO: 2
(b) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 2 and encodes a protein having tannase activity Recombinant DNA in which the tannase gene according to claim 2 or 3 is incorporated into vector DNA. A method for producing a protein, which comprises culturing a microorganism belonging to the genus Aspergillus and containing the recombinant DNA according to claim 4 in a medium, and collecting a protein having tannase activity from the obtained culture.

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