JP2004000122A - Alkaline protease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an alkaline protease having high cleaning ability even to multiple stains and also having high secretory ability. <P>SOLUTION: The alkaline protease is such one that an amino acid residue at (a) position 65, (b) position 101, (c) position 273, (d) position 320, (e) position 359 or (f) position 387 of a specific amino acid sequence or at a position corresponding thereto is selected from the following amino acid residues: position(a): proline, position(b): asparagine, position(c): isoleucine, glycine and threonine, position(d): phenylalanine, valine, threonine, leucine, isoleucine and glycine, position(e): serine, leucine, valine, isoleucine and glutamine, position(f): alanine, lysine, glutamine, glutamic acid, arginine and histidine. <P>COPYRIGHT: (C)2004,JPO

Description

【００２６】
また、本発明アルカリプロテアーゼにおけるアミノ酸残基の（ａ）〜（ｆ）の選択は、酵素活性及び酵素特性が変化しない限り２個所以上が同時になされていていもよい。２箇所以上が同時になされた場合の好ましい具体例を以下に示す。尚、アミノ酸は３文字表記とし、「＋」は１箇所の置換に対し付加された置換を表し、「／」については表記したいずれのアミノ酸を使用しても良いことを示している。
【００２７】
二重置換体の例としては、Ｔｈｒ６５Ｐｒｏ＋Ｇｌｙ１０１Ａｓｎ、Ｔｈｒ６５Ｐｒｏ＋Ｖａｌ２７３（Ｉｌｅ／Ｇｌｙ／Ｔｈｒ）、Ｇｌｙ１０１Ａｓｎ＋Ｔｈｒ３５９（Ｓｅｒ／Ｌｅｕ／Ｖａｌ／Ｉｌｅ／Ｇｌｎ）、Ｖａｌ２７３（Ｉｌｅ／Ｇｌｙ／Ｔｈｒ）＋Ｔｙｒ３２０（Ｐｈｅ／Ｖａｌ／Ｔｈｒ／Ｉｅｕ／Ｉｌｅ／Ｇｌｙ）等が挙げられるが、Ｔｈｒ６５Ｐｒｏ＋Ｓｅｒ３８７Ａｌａ、Ｔｈｒ３５９Ｓｅｒ＋Ｓｅｒ３８７Ａｌａが特に好ましい。
【００２８】
三重変異体の例としては、Ｔｈｒ６５Ｐｒｏ＋Ｇｌｙ１０１Ａｓｎ＋Ｖａｌ２７３（Ｉｌｅ／Ｇｌｙ／Ｔｈｒ）、Ｔｙｒ３２０（Ｐｈｅ／Ｖａｌ／Ｔｈｒ／Ｉｅｕ／Ｉｌｅ／Ｇｌｙ）＋Ｖａｌ２７３（Ｉｌｅ／Ｇｌｙ／Ｔｈｒ）＋Ｓｅｒ３８７（Ａｌａ／Ｌｙｓ／Ｇｌｎ／Ｇｌｕ／Ａｒｇ／Ｈｉｓ）、Ｔｈｒ６５Ｐｒｏ＋Ｔｙｒ３２０（Ｐｈｅ／Ｖａｌ／Ｔｈｒ／Ｉｅｕ／Ｉｌｅ／Ｇｌｙ）＋Ｔｈｒ３５９（Ｓｅｒ／Ｌｅｕ／Ｖａｌ／Ｉｌｅ／Ｇｌｎ）等が挙げられるが、Ｔｈｒ６５Ｐｒｏ＋Ｇｌｙ１０１Ａｓｎ＋Ｓｅｒ３８７Ａｌａ、Ｔｈｒ６５Ｐｒｏ＋Ｖａｌ２７３Ｉｌｅ＋Ｔｙｒ３２０Ｐｈｅ、Ｔｈｒ６５Ｐｒｏ＋Ｔｙｒ３２０Ｐｈｅ＋Ｓｅｒ３８７Ａｌａ等が好ましく、Ｔｈｒ６５Ｐｒｏ＋Ｖａｌ２７３Ｉｌｅ＋Ｔｈｒ３５９Ｓｅｒ、Ｔｈｒ６５Ｐｒｏ＋Ｖａｌ２７３Ｉｌｅ＋Ｓｅｒ３８７Ａｌａ、Ｔｈｒ６５Ｐｒｏ＋Ｔｙｒ３２０Ｇｌｙ＋Ｓｅｒ３８７Ａｌａ等が特に好ましい。
【００２９】
四重変異体の例としては、Ｔｈｒ６５Ｐｒｏ＋Ｇｌｙ１０１Ａｓｎ＋Ｖａｌ２７３（Ｉｌｅ／Ｇｌｙ／Ｔｈｒ）＋Ｔｙｒ３２０（Ｐｈｅ／Ｖａｌ／Ｔｈｒ／Ｉｅｕ／Ｉｌｅ／Ｇｌｙ）、Ｔｈｒ６５Ｐｒｏ＋Ｔｙｒ３２０（Ｐｈｅ／Ｖａｌ／Ｔｈｒ／Ｉｅｕ／Ｉｌｅ／Ｇｌｙ）＋Ｔｈｒ３５９（Ｓｅｒ／Ｌｅｕ／Ｖａｌ／Ｉｌｅ／Ｇｌｎ）＋Ｓｅｒ３８７（Ａｌａ／Ｌｙｓ／Ｇｌｎ／Ｇｌｕ／Ａｒｇ／Ｈｉｓ）、Ｇｌｙ１０１Ａｓｎ＋Ｖａｌ２７３（Ｉｌｅ／Ｇｌｙ／Ｔｈｒ）＋Ｔｙｒ３２０（Ｐｈｅ／Ｖａｌ／Ｔｈｒ／Ｉｅｕ／Ｉｌｅ／Ｇｌｙ）＋Ｓｅｒ３８７（Ａｌａ／Ｌｙｓ／Ｇｌｎ／Ｇｌｕ／Ａｒｇ／Ｈｉｓ）等が挙げられるが、Ｔｈｒ６５Ｐｒｏ＋Ｖａｌ２７３Ｉｌｅ＋Ｔｈｒ３５９（Ｓｅｒ／Ｌｅｕ／Ｉｌｅ／Ｖａｌ／Ｔｈｒ）＋Ｓｅｒ３８７（Ｇｌｕ／Ａｌａ）、Ｔｈｒ６５Ｐｒｏ＋Ｖａｌ２７３Ｉｌｅ＋Ｔｙｒ３２０（Ｖａｌ／Ｌｅｕ／Ｐｈｅ／Ｔｈｒ）＋Ｓｅｒ３８７（Ａｌａ／Ｈｉｓ／Ｇｌｎ）等が好ましく、Ｔｈｒ６５Ｐｒｏ＋Ｖａｌ２７３Ｉｌｅ＋Ｔｈｒ３５９Ｓｅｒ＋Ｓｅｒ３８７（Ａｌａ／Ｌｙｓ）、Ｔｈｒ６５Ｐｒｏ＋Ｖａｌ２７３Ｉｌｅ＋Ｔｈｒ３５９Ｇｌｎ＋Ｓｅｒ３８７Ａｌａ、Ｔｈｒ６５Ｐｒｏ＋Ｖａｌ２７３Ｉｌｅ＋Ｔｙｒ３２０Ｐｈｅ＋Ｓｅｒ３８７（Ｇｌｎ／Ｌｙｓ）、Ｔｈｒ６５Ｐｒｏ＋Ｖａｌ２７３Ｉｌｅ＋Ｔｙｒ３２０Ｉｌｅ＋Ｓｅｒ３８７Ｇｌｎ等が好ましい。
さらに五重、六重変異体の各組合せでも良い。
【００３０】
本発明のアルカリプロテアーゼが変異体である場合、変異を施す前のアルカリプロテアーゼ（親アルカリプロテアーゼということがある）としては、「配列番号１で示されるアミノ酸配列を有するプロテアーゼ」又は上述した「他種アルカリプロテアーゼ」として示したものが該当し、これに目的部位の変異を施すことにより本発明のアルカリプロテアーゼが得られる。例えばプロテアーゼＫＰ４３の配列番号１で示されるアミノ酸配列の前記（ａ）〜（ｆ）より選ばれる位置のアミノ酸残基又は他種アルカリプロテアーゼのアミノ酸配列において当該位置に相当する位置のアミノ酸残基を他のアミノ酸残基に置換することにより得られる。
【００３１】
本発明アルカリプロテアーゼは、例えば以下の方法により得ることができる。すなわち、クローニングされた親アルカリプロテアーゼをコードする遺伝子に対して変異を施し、得られた変異遺伝子を用いて適当な宿主を形質転換し、当該組換え宿主を培養し、培養物から採取することにより得られる。親アルカリプロテアーゼをコードする遺伝子のクローニングは、一般的な遺伝子組換え技術を用いればよく、例えばＷＯ９９／１８２１８、ＷＯ９８／５６９２７記載の方法に従って行えばよい。
【００３２】
親アルカリプロテアーゼをコードする遺伝子の変異手段としては、一般的に行われているランダム変異や部異特異的変異の方法がいずれも採用できる。より具体的には、例えばＳｉｔｅ−Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｓｙｓｔｅｍ　Ｍｕｔａｎ−Ｓｕｐｅｒ　Ｅｘｐｒｅｓｓ　Ｋｍキット（Ｔａｋａｒａ）等を用いて行うことができる。また、リコンビナントＰＣＲ（ｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ）法（ＰＣＲ　ｐｒｏｔｏｃｏｌｓ，　Ａｃａｄｅｍｉｃ　ｐｒｅｓｓ，　Ｎｅｗ　Ｙｏｒｋ，　１９９０）を用いることによって、遺伝子の任意の配列を他の遺伝子の該任意の配列に相当する配列と置換することが可能である。
【００３３】
得られた変異遺伝子を用いた本発明プロテアーゼの生産方法としては、例えば当該変異遺伝子を安定に増幅できるＤＮＡベクターに連結させ宿主菌を形質転換する、或いは当該変異遺伝子を安定に維持できる宿主菌の染色体ＤＮＡ上に導入させる、等の方法が採用できる。この条件を満たす宿主としては例えばバチルス属細菌、大腸菌、カビ、酵母、放線菌等が挙げられ、これらの菌株を用い、資化性の炭素源、窒素源その他必須栄養素を含む培地に接種し、常法に従い培養すればよい。
【００３４】
かくして得られた培養液中からのアルカリプロテアーゼの採取、及び精製は、一般の酵素の採取、及び精製方法に準じて行うことができる。例えば、培養液を遠心分離、又は濾過することで菌体を除き、培養上清液から常法の精製手段により目的酵素を得る。このようにして得られる酵素液は、そのまま用いることもできるが、更に公知の方法により精製、結晶化、粉末化、または顆粒化することもできる。
【００３５】
得られたプロテアーゼは、酸化剤耐性を有し、高濃度の脂肪酸によるカゼイン分解活性の阻害を受けず、ＳＤＳ−ＰＡＧＥにより認められる分子量が４３，０００±２，０００であり、アルカリ性域で活性を有すると共に、形質転換体での分泌能が高い。
ここで、「分泌能が高い」とは、例えば親アルカリプロテアーゼと同一の条件下において（例えばポリペプトンＳ８％（ｗ／ｖ）、酵母エキス０．３％、マルトース１０％、硫酸マグネシウム７水和物０．０４％、リン酸２水素カリウム０．２％、無水炭酸ナトリウム１．５％、テトラサイクリン３０ｐｐｍから成る培地に植菌（ｖ／ｖ）し、３０℃、３日間振とう培養する）、生産したアルカリプロテアーゼ変異体について培養上清中のプロテアーゼ活性量、タンパク質量を測定した場合、一定量以上の活性量又はタンパク質量を示すことをいい、例えば親アルカリプロテアーゼの５％以上、望ましくは１０％以上、さらに望ましくは２０％以上の活性量又はタンパク質量の増大が認められることを意味する。特に、比活性等の変化が認められなければ活性量とタンパク量の比は、親アルカリプロテアーゼと変異アルカリプロテアーゼでは一定の値をとると考えられることから、活性量又はタンパク質量のどちらか一方を測定しても構わない。
【００３６】
従って、本発明のアルカリプロテアーゼは、各種洗剤組成物配合用酵素として有用である。
洗浄剤組成物中への本発明アルカリプロテアーゼの配合量は、当該プロテアーゼが活性を示す量であれば特に制限されないが、洗浄剤組成物１ｋｇ当たり０．１〜５０００ＰＵが配合できるが、経済性等を考慮し、５００ＰＵ以下が好ましい。
【００３７】
本発明の洗浄剤組成物には、本発明のアルカリプロテアーゼ以外に様々な酵素を併用することもできる。例えば、加水分解酵素、酸化酵素、還元酵素、トランスフェラーゼ、リアーゼ、イソメラーゼ、リガーゼ、シンテターゼ等である。このうち、本発明以外のプロテアーゼ、セルラーゼ、ケラチナーゼ、エステラーゼ、クチナーゼ、アミラーゼ、リパーゼ、プルラナーゼ、ペクチナーゼ、マンナナーゼ、グルコシダーゼ、グルカナーゼ、コレステロールオキシダーゼ、ペルオキシダーゼ、ラッカーゼ等が好ましく、特にプロテアーゼ、セルラーゼ、アミラーゼ、リパーゼが好ましい。プロテアーゼとしては市販のアルカラーゼ（登録商標；ノボザイムズ社）、エスペラーゼ（登録商標；ノボザイムズ社）、サビナーゼ（登録商標；ノボザイムズ社）、エバラーゼ（登録商標；ノボザイムズ社）、カンナーゼ（登録商標；ノボザイムズ社）、プロペラーゼ（登録商標；ジェネンコア社）、プラフェクト（登録商標；ジェネンコア社）、ＫＡＰ（花王）等が挙げられる。セルラーゼとしてはセルザイム（登録商標；ノボザイムズ社）、ケアザイム（登録商標；ノボザイムズ社）、またＫＡＣ、特開平１０−３１３８５９号公報記載のバチルス・エスピーＫＳＭ−Ｓ２３７株が生産するアルカリセルラーゼ、特願２００２−１１６５５３号記載の変異アルカリセルラーゼ（以上、花王）等が挙げられる。アミラーゼとしてはターマミル（登録商標；ノボザイムズ社）、デュラミル（登録商標；ノボザイムズ社）、プラスター（登録商標；ジェネンコア社）、ＫＡＭ（花王）等が挙げられる。リパーゼとしてはリポラーゼ（登録商標；ノボザイムズ社）、リポラーゼウルトラ（登録商標；ノボザイムズ社）等が挙げられる。
【００３８】
洗浄剤組成物中で本発明のアルカリプロテアーゼ以外のプロテアーゼを併用する場合の配合量は、洗浄剤組成物１ｋｇ当たり０．１〜５００ＰＵが好ましい。また、セルラーゼを併用する場合は、特開平１０−３１３８５９号公報の段落〔００２０〕に記載の酵素活性測定方法より決定される単位（Ｕ）に基づき、洗浄剤組成物１ｋｇ当たり３００〜３００００００ＫＵが好ましく、アミラーゼを併用する場合は、特開平１１−４３６９０号公報の段落〔００４０〕記載のアミラーゼ活性測定方法より決定される単位（ＩＵ）に基づき、洗浄剤組成物１ｋｇ当たり５０〜５０００００ＩＵが好ましい。さらにリパーゼを併用する場合は、特表平８−５０００１３号公報の実施例１記載のリパーゼ活性測定方法より決定される単位（ＬＵ）づき、洗浄剤組成物１ｋｇ当たり１００００〜１００００００ＬＵが好ましい。
【００３９】
本発明の洗浄剤組成物には公知の洗浄剤成分を配合することができ、当該公知の洗浄剤成分としては、例えば次のものが挙げられる。
（１）界面活性剤
界面活性剤は洗浄剤組成物中０．５〜６０質量％配合され、特に粉末状洗浄剤組成物については１０〜４５質量％、液体洗浄剤組成物については２０〜５０質量％配合することが好ましい。また本発明洗浄剤組成物が漂白剤、または自動食器洗浄機用洗剤である場合、界面活性剤は一般に１〜１０質量％、好ましくは１〜５質量％配合される。
本発明洗浄剤組成物に用いられる界面活性剤としては、陰イオン性界面活性剤、非イオン性界面活性剤、両性界面活性剤、陽イオン性界面活性剤の１種または組み合わせを挙げることができるが、好ましくは陰イオン性界面活性剤、非イオン性界面活性剤である。
陰イオン性界面活性剤としては、炭素数１０〜１８のアルコールの硫酸エステル塩、炭素数８〜２０のアルコールのアルコキシル化物の硫酸エステル塩、アルキルベンゼンスルホン酸塩、パラフィンスルホン酸塩、α−オレフィンスルホン酸塩、α−スルホ脂肪酸塩、α−スルホ脂肪酸アルキルエステル塩又は脂肪酸塩が好ましい。本発明では特に、アルキル鎖の炭素数が１０〜１４の、より好ましくは１２〜１４の直鎖アルキルベンゼンスルホン酸塩が好ましく、対イオンとしては、アルカリ金属塩やアミン類が好ましく、特にナトリウム及び／又はカリウム、モノエタノールアミン、ジエタノールアミンが好ましい。
非イオン性界面活性剤としては、ポリオキシアルキレンアルキル（炭素数８〜２０）エーテル、アルキルポリグリコシド、ポリオキシアルキレンアルキル（炭素数８〜２０）フェニルエーテル、ポリオキシアルキレンソルビタン脂肪酸（炭素数８〜２２）エステル、ポリオキシアルキレングリコール脂肪酸（炭素数８〜２２）エステル、ポリオキシエチレンポリオキシプロピレンブロックポリマーが好ましい。特に、非イオン性界面活性剤としては、炭素数１０〜１８のアルコールにエチレンオキシドやプロピレンオキシド等のアルキレンオキシドを４〜２０モル付加した〔ＨＬＢ値（グリフィン法で算出）が１０．５〜１５．０、好ましくは１１．０〜１４．５であるような〕ポリオキシアルキレンアルキルエーテルが好ましい。
【００４０】
（２）二価金属イオン補捉剤
二価金属イオン補捉剤は０．０１〜５０質量％、好ましくは５〜４０質量％配合される。本発明洗浄剤組成物に用いられる二価金属イオン補捉剤としては、トリポリリン酸塩、ピロリン酸塩、オルソリン酸塩等の縮合リン酸塩、ゼオライト等のアルミノケイ酸塩、合成層状結晶性ケイ酸塩、ニトリロ三酢酸塩、エチレンジアミン四酢酸塩、クエン酸塩、イソクエン酸塩、ポリアセタールカルボン酸塩等が挙げられる。このうち結晶性アルミノケイ酸塩（合成ゼオライト）が特に好ましく、Ａ型、Ｘ型、Ｐ型ゼオライトのうち、Ａ型が特に好ましい。合成ゼオライトは、平均一次粒径０．１〜１０μｍ、特に０．１〜５μｍのものが好適に使用される。
【００４１】
（３）アルカリ剤
アルカリ剤は０．０１〜８０質量％、好ましくは１〜４０質量％配合される。粉末洗剤の場合、デンス灰や軽灰と総称される炭酸ナトリウム等のアルカリ金属炭酸塩、並びにＪＩＳ１号、２号、３号等の非晶質のアルカリ金属珪酸塩が挙げられる。これら無機性のアルカリ剤は洗剤乾燥時に、粒子の骨格形成において効果的であり、比較的硬く、流動性に優れた洗剤を得ることができる。これら以外のアルカリとしてはセスキ炭酸ナトリウム、炭酸水素ナトリウム等が挙げられ、またトリポリリン酸塩等のリン酸塩もアルカリ剤としての作用を有する。また、液体洗剤に使用されるアルカリ剤としては、上記アルカリ剤の他に水酸化ナトリウム、並びにモノ、ジ又はトリエタノールアミンを使用することができ、活性剤の対イオンとしても使用できる
【００４２】
（４）再汚染防止剤
再汚染防止剤は０．００１〜１０質量％、好ましくは１〜５質量％配合される。本発明洗浄剤組成物に用いられる再汚染防止剤としてはポリエチレングリコール、カルボン酸系ポリマー、ポリビニルアルコール、ポリビニルピロリドン等が挙げられる。このうちカルボン酸系ポリマーは再汚染防止能の他、金属イオンを捕捉する機能、固体粒子汚れを衣料から洗濯浴中へ分散させる作用がある。カルボン酸系ポリマーはアクリル酸、メタクリル酸、イタコン酸等のホモポリマーないしコポリマーであり、コポリマーとしては上記モノマーとマレイン酸の共重合したものが好適であり、分子量が数千〜１０万のものが好ましい。上記カルボン酸系ポリマー以外に、ポリグリシジル酸塩等のポリマー、カルボキシメチルセルロース等のセルロース誘導体、並びにポリアスパラギン酸等のアミノカルボン酸系のポリマーも金属イオン捕捉剤、分散剤及び再汚染防止能を有するので好ましい。
【００４３】
（５）漂白剤
例えば過酸化水素、過炭酸塩等の漂白剤は１〜１０質量％配合するのが好ましい。漂白剤を使用するときは、テトラアセチルエチレンジアミン（ＴＡＥＤ）や特開平６−３１６７００号公報記載等の漂白活性化剤（アクチベーター）を０．０１〜１０質量％配合することができる。
【００４４】
（６）蛍光剤
本発明洗浄剤組成物に用いられる蛍光剤としてはビフェニル型蛍光剤（例えばチノパールＣＢＳ−Ｘ等）やスチルベン型蛍光剤（例えばＤＭ型蛍光染料等）が挙げられる。蛍光剤は０．００１〜２質量％配合するのが好ましい。
【００４５】
（７）その他の成分
本発明品洗浄剤組成物には、衣料用洗剤の分野で公知のビルダー、柔軟化剤、還元剤（亜硫酸塩等）、抑泡剤（シリコーン等）、香料、その他の添加剤を含有させることができる。
【００４６】
本発明の洗浄剤組成物は、上記方法で得られた本発明アルカリプロテアーゼ及び上記公知の洗浄成分を組み合わせて常法に従い製造することができる。洗剤の形態は用途に応じて選択することができ、例えば液体、粉体、顆粒、ペースト、固形等にすることができる。
かくして得られる本洗浄剤組成物は、衣料洗浄剤、漂白剤、硬質表面洗浄用洗浄剤、排水管洗浄剤、義歯洗浄剤、医療器具用の殺菌洗浄剤等として使用することができる。
【００４７】
【実施例】
［プロテアーゼ活性測定法−カゼイン法］
カゼイン１％（ｗ／ｖ）を含む５０ｍＭホウ酸緩衝液（ｐＨ１０．５）１．０ｍＬを３０℃で５分間保温した後、０．１ｍＬの酵素溶液を加え、１５分間反応を行う。反応停止液（０．１１Ｍトリクロロ酢酸−０．２２Ｍ酢酸ナトリウム−０．３３Ｍ酢酸）を２．０ｍＬ加え、室温で３０分間放置した後、濾過を行い、濾液中の酸可溶性タンパク質をＬｏｗｒｙらの方法の変法により定量した。すなわち、０．５ｍＬの濾液にアルカリ性銅溶液（１％酒石酸ナトリウム・カリウム：１％硫酸銅・５水和物：２％炭酸ナトリウム・０．１Ｎ水酸化ナトリウム＝１：１：１００）を２．５ｍＬ加え、室温で１０分間放置後、フェノール溶液［フェノール試薬（関東化学）を蒸留水にて２倍希釈したもの］を０．２５ｍＬ添加し、３０℃で３０分間保温した。その後、６６０ｎｍにおける吸光度を測定した。プロテアーゼ１単位（１ＰＵ）は、上記反応条件で１分間に１ｍｍｏｌのチロシンに相当する酸可溶性タンパク質分解物を遊離させるのに必要な酵素量とした。
【００４８】
実施例１
バチルス　エスピーＫＳＭ−ＫＰ４３株由来のアルカリプロテアーゼ構造遺伝子の終止コドンまでを含む約２．０ｋｂの範囲に対しランダム変異を与えた。ランダム変異導入法としてはＰＣＲ中の塩基の取り込みエラーを利用するために、ＤＮＡポリメラーゼとしてエラー修復能が無いＴａｑポリメラーゼ：Ｔａｋａｒａ　Ｔａｑ（Ｔａｋａｒａ）を用いた。まず、上記約２．０ｋｂのＤＮＡを増幅できるプライマー１（５’−ＡＡＡＴＧＧＡＴＣＣＧＴＧＡＧＧＡＧＧＧＡＡＣＣＧＡＡＴＧＡＧＡＡＡＧＡＡＧＡＡＡＡＡＧＧＴＧ−３’、配列番号２）及びプライマー２（５’−ＡＴＡＴＴＣＴＡＧＡＣＧＡＴＴＡＣＣＡＴＡＴＴＡＡＴＴＣＣＴＣＴＡＣＣＣ−３’、配列番号３）を用いＰＣＲを行った。プライマー１はセンス鎖の５’末端側にＢａｍＨＩリンカーを、プライマー２はアンチセンス鎖の５’末端側にＸｂａＩリンカーを付与した。反応系として、鋳型ＤＮＡ１０ｎｇ、各プライマーを１０ｐｍｏｌ、各ｄＮＴＰを２０ｎｍｏｌ、Ｔａｋａｒａ　Ｔａｑ添付反応バッファー１０μＬ、及びＴａｑポリメラーゼ２．５Ｕを含有する１００μＬの系とした。ＰＣＲ条件は９４℃で２分間鋳型ＤＮＡを変性させた後、９４℃で１分間、５５℃で１分間、７２℃で２分間を１サイクルとし、これを３０サイクル行った。ＰＣＲ産物をＰＣＲ　ｐｒｏｄｕｃｔ　ｐｕｒｉｆｉｃａｔｉｏｎ　ｋｉｔ（ロッシュ）にて精製し、１００μＬの滅菌水で溶出した。次に溶出液１μＬを鋳型ＤＮＡとして、２回目のＰＣＲを行い、得られたＰＣＲ産物を精製し、以後の実験に供した。
【００４９】
増幅した約２．０ｋｂのＤＮＡ断片の末端制限酵素リンカーを、ＢａｍＨＩ、ＸｂａＩ（ロッシュ）により切断した。増幅ＤＮＡを組み込むべき発現ベクターとしてはバチルス属細菌内で複製可能であるｐＨＡ６４（特願平８−３２３０５０：プロモーター６４の下流にＢａｍＨＩ、ＸｂａＩサイトを有する）を用いた。ＢａｍＨＩ、ＸｂａＩ処理した増幅ＤＮＡ断片及び同じくＢａｍＨＩ、ＸｂａＩ処理したｐＨＡ６４を混合した後、Ｌｉｇａｔｉｏｎ　Ｈｉｇｈ（東洋紡）により、リガーゼ反応を行った。エタノール沈殿により、リガーゼ反応液からＤＮＡを回収し，以後の形質転換用のＤＮＡとした。
【００５０】
形質転換すべき宿主としてバチルスエスピーＫＳＭ−ＫＰ４３（以後ＫＰ−４３株と略す）を用いた。形質転換法はエレクトロポレーション法により行い、ＳＳＨ−１０（島津製作所）及びジーンパルサーキュベット（バイオラッド）を用い形質転換を行った。
【００５１】
ＫＰ４３株の形質転換体をスキムミルク含有アルカリ寒天培地［スキムミルク（ディフコ）１％（ｗ／ｖ）、バクトトリプトン（ディフコ）１％、酵母エキス（ディフコ）０．５％、塩化ナトリウム０．５％、寒天１．５％、無水炭酸ナトリウム０．０５％、テトラサイクリン１５ｐｐｍ］に生育させ、ハローの形成状況により、プロテアーゼ遺伝子導入の有無を判定した。
プロテアーゼ遺伝子がｐＨＡ６４に挿入されたプラスミドを保持している形質転換されたＫＰ４３株を選抜し、以後の培養に供した。
【００５２】
各形質転換体について単集落分離、ハロー形成の確認を行い、試験管中の５ｍＬ種母培地［ポリペプトンＳ（日本製薬）６．０％（ｗ／ｖ）、酵母エキス０．１％、マルトース１．０％、硫酸マグネシウム７水和物０．０２％、リン酸２水素カリウム０．１％、無水炭酸ナトリウム０．３％、テトラサイクリン３０ｐｐｍ］に植菌し、３０℃、３２０ｒｐｍで一晩前培養した。この種母培養液を５００ｍＬ容坂口フラスコ中の２０ｍＬ主培地［ポリペプトンＳ８％（ｗ／ｖ）、酵母エキス０．３％、マルトース１０％、硫酸マグネシウム７水和物０．０４％、リン酸２水素カリウム０．２％、無水炭酸ナトリウム１．５％、テトラサイクリン３０ｐｐｍ］に１％植菌（ｖ／ｖ）し、３０℃、１２１ｒｐｍで３日間培養した。得られた培養液を遠心分離し、培養上清中のプロテアーゼ活性を測定した。プロテアーゼ活性はカゼイン法により、タンパク質量はプロテインアッセイキット（和光純薬）を用いて測定した。野生型酵素遺伝子を有する形質転換体を同条件で培養した場合の培養上清の値と比較することにより、プロテアーゼ活性の向上が認められた変異プロテアーゼ遺伝子を選抜した。尚、培養上清中のタンパク質量はプロテアーゼ活性とほぼ比例して増加していることから、得られた変異体はタンパク質の分泌量が向上するのに必要な変異が導入されたことが、示唆された。
【００５３】
選抜された形質転換体からＨｉｇｈ　ｐｕｒｅ　ｐｌａｓｍｉｄ　ｉｓｏｌａｔｉｏｎ　ｋｉｔ（ロッシュ）を用いプラスミドを回収し、塩基配列を決定した。プラスミドＤＮＡ３００ｎｇを鋳型として、プライマーとＢｉｇ　Ｄｙｅ　ＤＮＡ　Ｓｅｑｕｅｎｃｉｎｇ　ｋｉｔ（アプライドバイオシステム）を用いて２０μＬの反応系でＰＣＲを行い、ＤＮＡ　Ｓｅｑｕｅｎｃｅｒ　３７７型（アプライドバイオシステム）を用いた解析に供した。
【００５４】
その結果、プロテアーゼ活性が向上した変異体は６５位のスレオニンがプロリン、１０１位のグリシンがアスパラギン、２７３位のバリンがイソロイシン、３２０位のチロシンがフェニルアラニン、３５９位のスレオニンがセリン、３８７位のセリンがアラニンにそれぞれ置換されており、これにより約５％のプロテアーゼ活性の向上が認められた（表２）。
【００５５】
更に上記変異部位を組合わせることにより、プロテアーゼ活性の向上を検討した。部位特異的変異の手段としては、Ｓｉｔｅ−Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　Ｓｙｓｔｅｍ　Ｍｕｔａｎ−Ｓｕｐｅｒ　Ｅｘｐｒｅｓｓ　Ｋｍキットを用い、以下のプライマーにより、変異部位の組み合わせを検討した。プライマー３：６５位のスレオニン（Ｔ）をプロリン（Ｐ）に置換する（５’−ＡＡＴＧＣＣＡＡＴＧＡＴＣＣＧＡＡＴＧＧＴＣＡＴＧ−３’、配列番号４）
プライマー４：１０１位のグリシン（Ｇ）をアスパラギン（Ｎ）に置換する（５’−ＴＣＴＡＴＣＡＴＧＧＡＴＡＧＣＡＡＴＧＧＧＧＧＡＣＴＴＧＧＡＧＧ−３’、配列番号５）プライマー５：２７３位のバリン（Ｖ）をイソロイシン（Ｉ）に置換する（５’−ＣＴＴＣＧＴＧＡＧＣＡＴＴＴＴＡＴＣＡＡＡＡＡＣＡＧＡＧＧＣＡＴＣ−３’、配列番号６）プライマー６：３２０位のチロシン（Ｙ）をフェニルアラニン（Ｆ）に置換する（５’−ＡＡＣＧＴＴＧＣＣＴＴＴＧＴＧＡＡＣＧＡＧＴＣＣ−３’、配列番号７）
プライマー７：３５９位のスレオニン（Ｔ）をセリン（Ｓ）に置換する（５’−ＧＣＧＡＧＣＡＣＡＴＣＴＧＣＴＴＣＣＧＴＡＡＣＧ−３’、配列番号８）
プライマー８：３８７位のセリン（Ｓ）をアラニン（Ａ）に置換する（５’−ＴＧＡＣＴＴＴＡＣＴＧＣＧＣＣＡＴＡＣＡＡＴＧＡＴＡＡＣ−３’、配列番号９）
【００５６】
変異導入用鋳型プラスミドの作製は、カナマイシン選択用アンバー変異マーカーを有するｐＫＦ１８ｋのマルチクローニングサイト中のＢａｍＨＩ、ＸｂａＩサイトに、前記スクリーニングで得られた変異プロテアーゼ遺伝子を導入することにより構築した。
【００５７】
部位特異的変異導入用ＰＣＲにはＴａｋａｒａ　ＬＡ　Ｔａｑ（Ｔａｋａｒａ）を用いた。５’末端をリン酸化したセレクションプライマー（Ｍｕｔａｎ−Ｓｕｐｅｒ　Ｅｘｐｒｅｓｓ　Ｋｍ　キット添付）及びプライマー３〜８の各変異導入プライマーを各々５ｐｍｏｌ及び鋳型プラスミド１０ｎｇを用い変異導入ＰＣＲを行った。反応条件は９４℃で２分間鋳型ＤＮＡを変性させた後、９４℃で１分間、５５℃で１分間、７２℃で４分間を１サイクルとし、これを３０サイクル行った。得られたＰＣＲ産物を用い大腸菌ＭＶ１１８４株を形質転換することにより、変異プラスミドを得た。得られた変異プラスミドは先述の塩基配列決定法に従い変異部位を確認した。
【００５８】
部位特異的変異により変異導入されたプロテアーゼ遺伝子をｐＨＡ６４に導入し、ＫＰ−４３株を形質転換し、先述の条件で培養することで、野生型酵素に比べプロテアーゼ活性が更に増大する変異部位の組み合せを検討した。
【００５９】
その結果、Ｔ６５Ｐ＋Ｓ３８７Ａ、Ｔ３５９Ｓ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｙ３２０Ｆ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｔ３５９Ｓ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｙ３２０Ｆ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｇ１０１Ｎ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｔ３５９Ｓ＋Ｓ３８７Ａの組み合わせにおいて１０〜３０％のプロテアーゼ活性の向上が認められた（変異部位の組み合わせは＋で示した。表１）。
【００６０】
更に以下のプライマーを使用し、６５位、１０１位、２７３位、３２０位、３５９位、３８７位のアミノ酸を任意のアミノ酸に置換し、それぞれの位置のアミノ酸が上記置換アミノ酸とは別のアミノ酸に置換が可能か否かを検討し、更にその組み合わせを検討した。
プライマー９：６５位のスレオニン（Ｔ）を任意のアミノ酸（Ｘ）に置換する（５’−ＣＧＡＡＴＡＡＴＧＣＣＡＡＴＧＡＴＮＮＮＡＡＴＧＧＴＣＡＴＧＧＴＡＣＧＣ−３’、配列番号１０）
プライマー１０：１０１位のグリシン（Ｇ）を任意のアミノ酸（Ｘ）に置換する（５’−ＣＴＡＴＣＡＴＧＧＡＴＡＧＣＮＮＮＧＧＧＧＧＡＣＴＴＧＧＡＧＧ−３’、配列番号１１）
プライマー１１：２７３位のバリン（Ｖ）を任意のアミノ酸（Ｘ）に置換する（５’−ＣＧＴＧＡＧＣＡＴＴＴＴＮＮＮＡＡＡＡＡＣＡＧＡＧＧＣＡＴＣＡＣＡＣＣ−３’、配列番号１２）
プライマー１２：３２０位のチロシン（Ｙ）を任意のアミノ酸（Ｘ）に置換する（５’−ＣＣＣＴＧＡＡＣＧＴＴＧＣＣＮＮＮＧＴＧＡＡＣＧＡＧＴＣＣ−３’、配列番号１３）
プライマー１３：３５９位のスレオニン（Ｔ）を任意のアミノ酸（Ｘ）に置換する（５’−ＣＣＣＣＴＧＣＧＡＧＣＡＣＡＮＮＮＧＣＴＴＣＣＧＴＡＡＣＧＣ−３’、配列番号１４）
プライマー１４：３８７位のセリン（Ｓ）を任意のアミノ酸（Ｘ）に置換する（５’−ＧＧＡＡＡＴＧＡＣＴＴＴＡＣＴＮＮＮＣＣＡＴＡＣＡＡＴＧＡＴＡＡＣＴＧＧ−３’、配列番号１５）
【００６１】
その結果、２７３位のバリンはイソロイシン以外にグリシン、スレオニンへの置換、３２０位のチロシンはフェニルアラニン以外にバリン、スレオニン、ロイシン、イソロイシン、グリシンへの置換、３５９位のスレオニンはセリン以外にロイシン、バリン、イソロイシン、グルタミンへの置換、３８７位のセリンはアラニン以外にリジン、グルタミン、グルタミン酸、アルギニン、ヒスチジンへの置換により、野生型と比べ酵素の分泌が向上していることが認められ、それぞれの位置で上記アミノ酸置換が可能であることが確認された。
【００６２】
その組み合わせの一部の結果を示すと、Ｔ６５Ｐ＋Ｙ３２０Ｆ＋Ｓ３８７Ｅ、Ｔ６５Ｐ＋Ｙ３２０Ｇ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｔ３５９Ｓ＋Ｓ３８７Ｅ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｔ３５９Ｌ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｔ３５９Ｉ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｖ２７３Ｇ＋Ｔ３５９Ｓ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｔ３５９Ｓ＋Ｓ３８７Ｋ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｔ３５９Ｖ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｔ３５９Ｑ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｙ３２０Ｔ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｙ３２０Ｆ＋Ｓ３８７Ｅ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｙ３２０Ｆ＋Ｓ３８７Ｋ、Ｔ６５Ｐ＋Ｖ２７３Ｔ＋Ｙ３２０Ｆ＋Ｓ３８７Ａ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｙ３２０Ｌ＋Ｓ３８７Ｈ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｙ３２０Ｖ＋Ｓ３８７Ｑ、Ｔ６５Ｐ＋Ｖ２７３Ｉ＋Ｙ３２０Ｉ＋Ｓ３８７Ｑの組み合わせにおいて１０〜３０％のプロテアーゼ活性の向上が認められた（表２）。
【００６３】
【表２】

【００６４】
上記変異部位の組み合わせにより得られる、アルカリプロテアーゼは形質転換体での酵素の分泌を向上させる以外は親アルカリプロテアーゼの特性、すなわち、酸化剤耐性を有し、高濃度の脂肪酸によるカゼイン分解活性の阻害を受けず、ＳＤＳ−ＰＡＧＥにより認められる分子量が４３，０００±２，０００であり、アルカリ性域で活性を有する性質を保持していることを確認した。
実施例２
（１）洗剤の調製
撹拌翼を有した１ｍ^３の混合槽に水４６５ｋｇを加え、水温が５５℃に達した後に４０％（ｗ／ｖ）ポリアクリル酸ナトリウム水溶液１３５ｋｇを添加した。１５分間撹拌した後に、炭酸ナトリウム１２０ｋｇ、硫酸ナトリウム６０ｋｇ、亜硫酸ナトリウム９ｋｇ、蛍光染料３ｋｇを添加した。更に１５分間撹拌した後に、ゼオライト３００ｋｇを添加し、３０分間撹拌して均質なスラリーを得た（スラリー中の水分は５０質量％）。このスラリーを噴霧乾燥塔の塔頂付近に設置した圧力噴霧ノズルから噴霧することでベース顆粒を得た（噴霧乾燥塔に供給する高温ガスは塔下部より温度が２２５℃で供給し、塔頂より１０５℃で排出）。次にレディゲミキサー（松阪技研（株）製、容量２０Ｌ、ジャケット付）に上記ベース顆粒１００質量部を投入し、主軸（１５０ｒｐｍ）の攪拌下、非イオン性界面活性剤２０質量部、直鎖アルキル（炭素数１０〜１３）ベンゼンスルホン酸ナトリウム２２質量部、脂肪酸（炭素数１４〜１８）ナトリウム４質量部、ポリエチレングリコール２質量部、水４質量部の混合液を３分間で投入し、その後５分間攪拌を行った。更にこのミキサーに結晶性ケイ酸ナトリウム２０質量部とゼオライト１０質量部を投入し、表面被覆を行い洗剤ベースを得た。
洗剤ベース９９質量％に本発明プロテアーゼ粒子０．５質量％、及び香料０．５質量％を混合して最終粒状洗剤Ａを得た。
【００６５】
（２）使用した原料
非イオン性界面活性剤：エチレンオキサイド平均付加モル数が８．５のエマルゲン１０８ＫＭ（花王（株）製）
ポリアクリル酸ナトリウム水溶液：平均分子量１００００（特公平２−２４２８３号公報の実施例に記載の方法に従って製造した）
炭酸ナトリウム：デンス灰（セントラル硝子（株）製）
ゼオライト：平均粒径が３．５μｍのゼオライト４Ａ型（東ソー（株）製）
ポリエチレングリコール：Ｋ−ＰＥＧ６０００（平均分子量８５００，花王（株）製）
結晶性ケイ酸ナトリウム：粉末ＳＫＳ−６（ヘキストトクヤマ（株）製）
本発明プロテアーゼ粒子：表２記載の本発明アルカリプロテアーゼの各精製標品から特開昭６２−２５７９９０号公報の実施例１に記載の方法に基づき調製した造粒物（６ＰＵ／ｇ）
蛍光染料：チノパールＣＢＳ−Ｘ（チバガイギー社製）
【００６６】
実施例３
（１）洗剤の調製
まず固形分５０質量％のスラリーを熱風温度２５０℃で噴霧乾燥し、ポリアクリル酸ナトリウム（質量平均分子量１００００）７質量％、炭酸ナトリウム２６質量％、硫酸ナトリウム２０質量％、塩化ナトリウム６質量％、蛍光染料０．５質量％、ゼオライト４０質量％、水０．５質量％のベース顆粒を得た。
次にレディゲミキサー（松阪技研（株）製、容量２０Ｌ、ジャケット付）に上記ベース顆粒１００質量部を投入し、主軸（１５０ｒｐｍ）の攪拌下、非イオン性界面活性剤２０質量部、直鎖アルキル（炭素数１０〜１３）ベンゼンスルホン酸ナトリウム２２質量部、脂肪酸（炭素数１４〜１８）ナトリウム４質量部、ポリエチレングリコール２質量部、水４質量部の混合液を３分間で投入し、その後５分間攪拌を行った。更にこのミキサーに結晶性ケイ酸ナトリウム２０質量部とゼオライト１０質量部を投入し、表面被覆を行い洗剤ベースを得た。
洗剤ベース９５質量％に漂白剤粒子２．８質量％、漂白活性剤粒子１．２質量％、本発明プロテアーゼ粒子０．５質量％、及び香料０．５質量％を混合して最終粒状洗剤Ｂを得た。
【００６７】
（２）使用した原料
非イオン性界面活性剤：エチレンオキサイド平均付加モル数が８．５のエマルゲン１０８ＫＭ（花王（株）製）
ポリアクリル酸ナトリウム水溶液：平均分子量１００００（特公平２−２４２８３号公報の実施例に記載の方法に従って製造した）
炭酸ナトリウム：デンス灰（セントラル硝子（株）製）
ゼオライト：平均粒径が３．５μｍのゼオライト４Ａ型（東ソー（株）製）
ポリエチレングリコール：Ｋ−ＰＥＧ６０００（平均分子量８５００，花王（株）製）
結晶性ケイ酸ナトリウム：ＳＫＳ−６（ヘキストトクヤマ（株）製）
本発明プロテアーゼ粒子：表２記載の本発明アルカリプロテアーゼの各精製標品から特開昭６２−２５７９９０号公報の実施例１に記載の方法に基づき調製した造粒物（６ＰＵ／ｇ）
蛍光染料：チノパールＣＢＳ−Ｘ（チバガイギー社製）
漂白剤粒子：炭酸ナトリウム・過酸化水素付加物（特開２０００−２５６６９９号公報の段落〔００１９〕記載の漂白剤粒子と同様にして得た）
漂白活性剤粒子：ラウロイルオキシベンゼンスルホン酸ナトリウムの造粒物（特開２０００−２５６６９９号公報の段落〔００１８〕記載の漂白活性剤粒子と同様にして得た）
【００６８】
実施例４
表３に示す液体洗浄剤組成物（洗剤Ｃ、及び洗剤Ｄ）を調製した。
【００６９】
【表３】

【００７０】
１）炭素数１２〜１４の２級アルコール由来のアルキル基を有するポリオキシエチレン（平均７モル付加）アルキルエーテル（ソフタノール７０、日本触媒化学工業製）
２）炭素数１２〜１４の２級アルコール由来のアルキル基を有するポリオキシエチレン（平均１２モル付加）アルキルエーテル（ソフタノール１２０、日本触媒化学工業製）
３）炭素数１０〜１４の直鎖第１級アルコールにＥＯを平均５モル、ＰＯを平均２モル、ＥＯを平均３モルの順にブロック付加させたもの
４）ポリオキシエチレンラウリルエーテル、ＥＯを平均８モル付加させたもの５）ポリオキシエチレンラウリルエーテル、ＥＯを平均１１．５モル付加させたもの
６）ナローレンジポリオキシエチレンアルキル（ｓｅｃ−Ｃ_１２／Ｃ_１３）エーテル
７）炭素数１０〜１４の直鎖アルキルベンゼンスルホン酸ナトリウム
８）アミド／エーテル変性シリコーンポリマー（東レ・ダウコーニングシリコーン（株）製、ＢＹ１６−９０６）
９）特開平１０−６０４７６号公報の１１頁６行〜１３行記載の方法で合成したフェノキシポリエチレングリコール、アクリル酸、マレイン酸共重合体（質量平均分子量１００００、固形分５１．２％）
１０）ペンテン／マレイン酸（５０／５０モル比）コポリマーのナトリウム塩（質量平均分子量７０００）
１１）表２記載の本発明アルカリプロテアーゼの各精製標品（１５ＰＵ／ｇ）
【００７１】
実施例５
下記の表４に示す組成のうち、過炭酸ナトリウムと炭酸ナトリウム（デンス灰）を攪拌混合しながら、ポリアクリル酸ナトリウム４０％水溶液、及び直鎖アルキルベンゼンスルホン酸ナトリウム、又は非イオン性界面活性剤、又はラウロイルオキシベンゼンスルホン酸ナトリウムを添加した。次いで特開昭６２―２５７９９０号公報の実施例１に記載の方法に基づき調製した本発明プロテアーゼ粒子を添加し、全体的に均一になる程度攪拌することにより、漂白剤を調製した。
【００７２】
【表４】

【００７３】
１）粒経５００〜７００μｍ
２）炭素数１２〜１４の直鎖アルキルベンゼンスルホン酸ナトリウム
３）ポリオキシエチレンアルキルエーテル（アルキル基の炭素数１２〜１４、ＥＯ平均付加モル数１２）
４）平均分子量８，０００
５）表２記載の本発明アルカリプロテアーゼの各精製標品から特開昭６２−２５７９９０号公報の実施例１に記載の方法に基づき調製した造粒物（６ＰＵ／ｇ）
【００７４】
実施例６
下記の表５に示す全自動食器洗浄機用洗浄剤組成物（洗剤Ｇ、及びＨ）を調製した。
【００７５】
【表５】

【００７６】
１）ポリオキシエチレン−ポリオキシプロピレン共重合体（平均分子量２，０００）
２）炭素数１２〜１４のｓｅｃ−アルコールのエチレンオキサイド７モル、及びプロピレンオキサイド８．５モル付加物
３）ＪＩＳ　２号珪酸ナトリウム
４）アクリル酸−マレイン酸共重合体
５）デュラミル６０Ｔ^（ＴＭ）（ノボザイムズ社製）
６）表２記載の本発明アルカリプロテアーゼの各精製標品から特開昭６２−２５７９９０号公報の実施例１に記載の方法に基づき調製した造粒物（６ＰＵ／ｇ）
【００７７】
実施例７
下記の表６に示す各成分を用い、硬質表面用洗浄剤組成物（洗剤Ｊ）を得た。
【００７８】
【表６】

【００７９】
１）ポリオキシエチレン（ＥＯＰ＝４）アルキル（Ｃ１２）エーテル硫酸エステルナトリウム
２）ポリオキシエチレン（ＥＯＰ＝８）アルキル（Ｃ１２）エーテル
３）アルキル（Ｃ１２）ポリグルコシド（縮合度１．３）
４）モノ長鎖第３級アルキル（Ｃ１２）ジメチルアミンオキシド
５）アルキル（Ｃ１２）ヒドロキシジメチルスルホベタイン
６）分子量１００００
７）表２記載の本発明アルカリプロテアーゼの各精製標品（１５ＰＵ／ｍＬ）
【００８０】
実施例８
前記洗剤Ａ（実施例２参照）を用いて下記表７記載の粒状洗剤を得た。
【００８１】
【表７】

【００８２】
１）表２記載の本発明アルカリプロテアーゼの各精製標品から特開昭６２−２５７９９０号公報の実施例１に記載の方法に基づき調製した造粒物（６ＰＵ／ｇ）
２）特開平５−２５４９２号公報に記載のプロテアーゼＫ−１６を特開昭６２−２５７９９０号公報の実施例１に記載の方法に基づき、５ＰＵ／ｇとしたもの
３）ＫＡＣ−５００（花王（株）製）
４）リポラーゼ１００Ｔ^（ＴＭ）（ノボザイムズ社製）
【００８３】
【発明の効果】
本発明によれば、高濃度の脂肪酸存在下でも活性を有し、蛋白質だけでなく皮脂等の混在する複合汚れに対しても優れた洗浄性を有すると共に、分泌能の高いアルカリプロテアーゼを提供できる。
【００８４】
【配列表】

【図面の簡単な説明】
【図１】配列番号１で示されるアミノ酸配列と８０％以上の相同性を有するアルカリプロテアーゼのアミノ酸配列を整列させた図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an alkaline protease useful as a detergent-containing enzyme and a gene encoding the same.
[0002]
Problems to be solved by the prior art and the invention
The use of proteases in the industrial field has a long history, and is very diverse, from detergents such as clothing detergents to fiber modifiers, leather treatment agents, cosmetics, bath agents, food modifiers or pharmaceuticals. Over. Among them, the most industrially produced proteases are detergent proteases, for example, Alcalase (registered trademark; Novozymes), Savinase (registered trademark; Novozymes), Maxacal (registered trademark; Genencor), Blapp ( (Registered trademark; Henkel) and protease K (Kao) are known.
[0003]
The purpose of incorporating protease into detergents is to decompose the protein-based dirt attached to clothing to lower molecular weight and promote solubilization by surfactants. Instead, it is a composite soil containing a plurality of components in which organic substances and inorganic substances are mixed, such as lipids and solid particles derived from sebum, and a detergent having high detergency for such composite soil has been desired.
[0004]
From such a viewpoint, the present inventors maintain a sufficient casein degrading activity even in the presence of a high concentration of fatty acids, and have a molecular weight of about 43, which has excellent detergency not only for proteins but also for complex soils including sebum and the like. Several 000 alkaline proteases were found, and a patent application was filed earlier (see Patent Document 1). Such alkaline protease group, unlike its conventionally known subtilisin, a serine protease derived from a bacterium belonging to the genus Bacillus, in its molecular weight, primary structure, enzymatic properties, particularly in that it has very strong oxidant resistance, Classification into a new subtilisin subfamily has been proposed (see Non-Patent Document 1).
[0005]
However, even if such a protease is produced on an industrial level, its production cannot be said to be sufficient, and an alkaline protease that is efficiently secreted into the medium has been required.
[0006]
On the other hand, attempts to secrete a large amount of the target protein (enzyme) include improving the host bacterium (producing bacterium) by mutation breeding, and modifying the gene encoding the enzyme or the gene that controls the enzyme to increase the amount of secretion. Although it is known, there is no example of modification of subtilisin that increases the secretion amount of the enzyme.
[0007]
Accordingly, an object of the present invention is to provide an alkaline protease having excellent detergency even for complex soils and having a high secretion ability.
[0008]
[Patent Document 1]
International Publication No. 99/18218 pamphlet
[Non-patent document 1]
Saeki et al., Biochem. Biophys. Res. Commun. , 279, (2000), 313-319.
[0009]
[Means for Solving the Problems]
The present inventors have searched for a new enzyme that can efficiently secrete into the medium while maintaining the properties of the alkaline protease, and found that a specific alkaline protease has a specific position at a specific position in the amino acid sequence. It has been found that amino acid residues are required.
[0010]
That is, the amino acid residue at (a) position 65, (b) position 101, (c) position 273, (d) position 320, (e) position 359, (f) position 387 or a position corresponding thereto in SEQ ID NO: 1 A group having the following amino acid residue;
(A) position: proline,
(B) location: asparagine,
(C) Position: isoleucine, glycine, threonine,
(D) position: phenylalanine, valine, threonine, leucine, isoleucine, glycine,
(E) Position: serine, leucine, valine, isoleucine, glutamine,
(F) Position: alanine, lysine, glutamine, glutamic acid, arginine, histidine,
And a gene encoding the same.
[0011]
The present invention also provides a vector containing the gene and a transformant containing the vector.
[0012]
The present invention also provides a detergent composition containing the alkaline protease.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
As described above, the alkaline protease of the present invention comprises (a) position 65, (b) position 101, (c) position 273, (d) position 320, (e) position 359, and (f) 387 of SEQ ID NO: 1. An amino acid residue at the position or a position corresponding thereto is the following amino acid residue;
(A) position: proline, (b) position: asparagine, (c) position: isoleucine, glycine, threonine, (d) position: phenylalanine, valine, threonine, leucine, isoleucine, glycine, (e) position: serine, leucine , Valine, isoleucine, glutamine, (f) position: alanine, lysine, glutamine, glutamic acid, arginine, histidine.
[0014]
That is, the alkaline protease of the present invention is an alkaline protease having an amino acid sequence represented by SEQ ID NO: 1 at an amino acid residue at a position selected from the above (a) to (f) or at the amino acid sequence of another kind of alkaline protease. It means a protease in which the amino acid residue at the corresponding position is a specific amino acid residue, and these may be a wild-type, a wild-type mutant, or an artificially mutated mutant.
[0015]
Here, the “other alkaline protease” may be a wild-type or wild-type mutant, has resistance to oxidants, and has a molecular weight of 43% by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE). 2,000 ± 2,000, and includes those having an amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 1. Particularly preferably, it has an amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 1, acts in an alkaline region of pH 8 or more, has oxidant resistance, and is treated at 50 ° C. and pH 10 for 10 minutes. An enzyme exhibiting a residual activity of at least 80%, which is inhibited by diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) and has a molecular weight of 43,000 ± 2,000 by SDS-PAGE. . Here, having the oxidizing agent resistance means that the alkaline protease has at least a residual activity after standing at 30 ° C. for 20 minutes in 20 mM Britton-Robinson buffer (pH 10) containing 50 mM hydrogen peroxide and 5 mM calcium chloride. It means that 50% or more is maintained.
[0016]
Here, the “alkali protease having the amino acid sequence represented by SEQ ID NO: 1” includes KP43 [derived from Bacillus sp. KSM-KP43 (FERM BP-6532), WO99 / 18218], and the “amino acid represented by SEQ ID NO: 1”. Examples of the “alkali protease having an amino acid sequence having 80% or more homology with the sequence” include protease KP9860 [derived from Bacillus sp. KSM-KP9860 (FERMBP-6534), WO 99/18218, GenBank Accession No. AB046403], protease E-1 [Bacillus No. D-6 (FERMP-1592), JP-A-49-71191, GenBank Accession No. AB046402], derived from protease Ya [Bacillus sp. Y (FERMBP-1029), JP-A-61-280268, GenBank Accession No. AB046404], derived from protease SD521 [Bacillus SD521 (FERMP-11162), JP-A-3-191781, GenBank Accession No. AB046405], protease A-1 [derived from NCIB12289, WO88 / 01293, GenBank Accession No. AB046406], protease A-2 [derived from NCIB12513, WO98 / 56927], a mutant in which position 46 of the amino acid sequence of SEQ ID NO: 1 is substituted with leucine, a mutant in which position 57 is substituted with alanine, position 103 is substituted with arginine , Lysine at position 107, lysine at position 124, alanine at position 136, alanine at position 193, and 195 at position 195. Mutants substituted with asparagine, glutamic acid, arginine, proline, threonine, valine, histidine, serine, lysine, glutamine, methionine, cysteine, alanine, aspartic acid, tryptophan, glycine and phenylalanine, respectively, and the 247th position with threonine and arginine, respectively Mutated mutant, mutant having valine at position 257, mutant having alanine at position 342, and double mutant having aspartic acid at position 66 and serine at position 264 (Japanese Patent Application No. No. 355166), a mutant in which position 84 of the amino acid sequence of SEQ ID NO: 1 was substituted with arginine, a mutant in which position 104 was substituted with proline, a mutant in which position 256 was substituted with alanine and serine, and position 369 was asparagine. Substituted mutant (Japanese Patent Application No. 13-114048), a mutant in which position 251 of the amino acid sequence of SEQ ID NO: 1 has been substituted with asparagine, threonine, isoleucine, valine, leucine and glutamine, respectively, and position 256 has serine and glutamine, respectively. Mutants substituted with asparagine, valine and alanine (Japanese Patent Application No. 13-329472) or this 80% or more in the amino acid sequence, preferably 87% or more, more preferably 90% or more, more preferably include alkaline protease having at least 95% homology.
The amino acid sequence homology is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985).
[0017]
In addition, as a method for specifying the “amino acid residue at the corresponding position”, for example, the amino acid sequences are compared using a known algorithm such as the Lippman-Person method, and conserved amino acid residues existing in the amino acid sequence of each alkaline protease are determined. This can be done by giving the groups maximum homology. By arranging the amino acid sequences of proteases by such a method, it is possible to determine the positions of homologous amino acid residues in the sequence of each protease, regardless of insertions or deletions in the amino acid sequence. Homologous positions are considered to be at the same position in the three-dimensional structure, and can be presumed to have a similar effect on the specific function of the protease of interest.
[0018]
That is, from FIG. 1 in which the amino acid sequences were aligned by the above method, (a) the amino acid residue at position 65 in SEQ ID NO: 1 is a threonine residue, and the amino acid residue at a position corresponding to that position is By using the method, for example, the threonine residue at position 65 in protease KP9860 can be specified. The amino acid residue is preferably a proline residue.
[0019]
(B) The amino acid residue at position 101 in SEQ ID NO: 1 is a glycine residue. Here, the amino acid residue at the position corresponding to that position is, for example, in protease E-1 by using the above method. Can be specified as the serine residue at position 100. The amino acid residue is preferably an asparagine residue.
[0020]
(C) The amino acid residue at position 273 of SEQ ID NO: 1 is a valine residue. Here, the amino acid residue at a position corresponding to that position is, for example, in protease A-2 by using the above method. Can be specified as the valine residue at position 272. The amino acid residue is preferably an isoleucine, glycine, or threonine residue, and particularly preferably an isoleucine residue.
[0021]
(D) The amino acid residue at position 320 of SEQ ID NO: 1 is a tyrosine residue. Here, the amino acid residue at the position corresponding to the position is determined by using the above-described method, for example, in protease SD-521. Can be specified as the tyrosine residue at position 319. The amino acid residue is preferably a phenylalanine, valine, threonine, leucine, isoleucine, or glycine residue, and particularly preferably a phenylalanine residue.
[0022]
(E) The amino acid residue at position 359 in SEQ ID NO: 1 is a threonine residue. Here, the amino acid residue at a position corresponding to that position is determined by using the above method, for example, 358 in protease Ya. Position threonine residue. The amino acid residue is preferably a serine, leucine, valine, isoleucine or glutamine residue, particularly preferably a serine residue.
[0023]
(F) The amino acid residue at position 387 of SEQ ID NO: 1 is a serine residue. Here, the amino acid residue at the position corresponding to the position is determined by using the above method, for example, in protease SD-521. Can be specified as the tyrosine residue at position 386. The amino acid residue is preferably an alanine, lysine, glutamine, glutamic acid, arginine, histidine residue, and particularly preferably an alanine residue.
[0024]
Positions corresponding to (a) position 65, (b) position 101, (c) position 273, (d) position 320, (e) position 359, and (f) position 387 of the amino acid sequence of protease KP43 (SEQ ID NO: 1). And specific examples of the amino acid residue are shown in Table 1 which are preferably used among the above-mentioned "other alkaline proteases".
[0025]
[Table 1]

[0026]
The selection of the amino acid residues (a) to (f) in the alkaline protease of the present invention may be performed at two or more sites at the same time as long as the enzyme activity and enzyme characteristics are not changed. Preferred examples when two or more points are made at the same time are shown below. Amino acids are represented by three letters, "+" represents substitution added to one substitution, and "/" indicates that any of the amino acids described may be used.
[0027]
Examples of the double substituent include Thr65Pro + Gly101Asn, Thr65Pro + Val273 (Ile / Gly / Thr), Gly101Asn + Thr359 (Ser / Leu / Val / Ile / Gln), and Val273 (Ile / Gly / ThrIhr / Thr / Thr / Thr / Thr / ThrThr / Thr / Thr / Thr / Th320). / Ile / Gly) and the like, but Thr65Pro + Ser387Ala and Thr359Ser + Ser387Ala are particularly preferable.
[0028]
Examples of the triple mutant, Thr65Pro + Gly101Asn + Val273 (Ile / Gly / Thr), Tyr320 (Phe / Val / Thr / Ieu / Ile / Gly) + Val273 (Ile / Gly / Thr) + Ser387 (Ala / Lys / Gln / Glu / Arg / His), Thr65Pro + Tyr320 (Phe / Val / Thr / Ieu / Ile / Gly) + Thr359 (Ser / Leu / Val / Ile / Gln) and others as listed, Thr65Pro + Gly101Asn + Ser387Ala, Thr65Pro + Val273Ile + Tyr320Phe, etc. Thr65Pro + Tyr320Phe + Ser387Ala preferably, Thr65Pro + Val273Ile + Thr359Ser Thr65Pro + Val273Ile + Ser387Ala, such as Thr65Pro + Tyr320Gly + Ser387Ala is particularly preferred.
[0029]
Examples of quadruple mutants include Thr65Pro + Gly101Asn + Val273 (Ile / Gly / Thr) + Tyr320 (Phe / Val / Thr / Ieu / Ile / Gly) and Thr65Pro + Tyr320 (Phe / Val / Thr / Gyr / Thr / Gle / Thr / Gr9). Leu / Val / Ile / Gln) + Ser387 (Ala / Lys / Gln / Glu / Arg / His), Gly101Asn + Val273 (Ile / Gly / Thr) + Tyr320 (Phe / Val / Thr / Ieu / Ile / Gly) + Ser / Gly + Ser3 + Ser387 (Ala / Lys / Gln / Glu / Arg / His) / Gln / Glu / Arg / His) and the like, but Thr65Pro + Val273Ile + Thr359 (Ser / Leu / Ile / Val / Thr) + er387 (Glu / Ala), and the like are preferred Thr65Pro + Val273Ile + Tyr320 (Val / Leu / Phe / Thr) + Ser387 (Ala / His / Gln), Thr65Pro + Val273Ile + Thr359Ser + Ser387 (Ala / Lys), Thr65Pro + Val273Ile + Thr359Gln + Ser387Ala, Thr65Pro + Val273Ile + Tyr320Phe + Ser387 (Gln / Lys), etc. Thr65Pro + Val273Ile + Tyr320Ile + Ser387Gln are preferred.
Furthermore, each combination of the fivefold and sixfold mutants may be used.
[0030]
When the alkaline protease of the present invention is a mutant, the alkaline protease before mutation (sometimes referred to as a parent alkaline protease) may be a “protease having the amino acid sequence represented by SEQ ID NO: 1” or the above “other species”. The alkaline protease of the present invention can be obtained by mutation of the target site. For example, the amino acid residue at the position selected from the above (a) to (f) of the amino acid sequence represented by SEQ ID NO: 1 of protease KP43 or the amino acid residue at the position corresponding to the position in the amino acid sequence of another alkaline protease is By substituting the amino acid residue with
[0031]
The alkaline protease of the present invention can be obtained, for example, by the following method. That is, by mutating the cloned gene encoding the parent alkaline protease, transforming an appropriate host using the obtained mutated gene, culturing the recombinant host, and collecting from the culture can get. Cloning of the gene encoding the parent alkaline protease may be performed by using a general gene recombination technique, for example, according to the method described in WO99 / 18218 and WO98 / 56927.
[0032]
As a means for mutating the gene encoding the parent alkaline protease, any commonly used method of random mutation or partial mutation can be employed. More specifically, it can be performed using, for example, a Site-Directed Mutagenesis System Mutan-Super Express Km kit (Takara) or the like. In addition, by using a recombinant PCR (polymerase chain reaction) method (PCR protocol, Academic Press, New York, 1990), it is possible to replace an arbitrary sequence of a gene with a sequence corresponding to the arbitrary sequence of another gene. It is possible.
[0033]
Examples of the method for producing the protease of the present invention using the obtained mutant gene include, for example, ligation to a DNA vector capable of stably amplifying the mutant gene, transformation of a host cell, or production of a host cell capable of stably maintaining the mutant gene. A method such as introduction into chromosomal DNA can be employed. Examples of hosts that satisfy this condition include Bacillus bacteria, Escherichia coli, mold, yeast, actinomycetes, and the like.Using these strains, assimilating carbon source, nitrogen source, and inoculating a medium containing other essential nutrients, Culture may be performed according to a conventional method.
[0034]
Collection and purification of the alkaline protease from the culture solution thus obtained can be performed in accordance with general methods for collecting and purifying enzymes. For example, cells are removed by centrifuging or filtering the culture solution, and the target enzyme is obtained from the culture supernatant by a conventional purification means. The enzyme solution thus obtained can be used as it is, or can be further purified, crystallized, powdered, or granulated by a known method.
[0035]
The obtained protease has resistance to oxidizing agents, is not inhibited by high-concentration fatty acids, and has a molecular weight recognized by SDS-PAGE of 43,000 ± 2,000. As well as high secretory ability in transformants.
Here, “high secretion ability” means, for example, under the same conditions as the alkali-protease (eg, polypeptone S 8% (w / v), yeast extract 0.3%, maltose 10%, magnesium sulfate heptahydrate) Inoculate (v / v) a medium consisting of 0.04%, potassium dihydrogen phosphate 0.2%, anhydrous sodium carbonate 1.5% and tetracycline 30ppm (v / v) and shake culture at 30 ° C for 3 days), production When the amount of protease activity and the amount of protein in the culture supernatant are measured for the alkali protease mutant thus obtained, it indicates that the activity or protein amount is a certain amount or more, for example, 5% or more, preferably 10% of the parent alkali protease. As described above, more preferably, an increase in the amount of activity or protein by 20% or more is recognized. In particular, unless a change in specific activity or the like is observed, the ratio between the amount of activity and the amount of protein is considered to take a constant value for the parent alkaline protease and the mutant alkaline protease. You may measure it.
[0036]
Therefore, the alkaline protease of the present invention is useful as an enzyme for blending various detergent compositions.
The blending amount of the alkaline protease of the present invention in the detergent composition is not particularly limited as long as the protease exhibits an activity, but 0.1 to 5000 PU can be blended per 1 kg of the detergent composition. In consideration of the above, 500 PU or less is preferable.
[0037]
Various enzymes other than the alkaline protease of the present invention can be used in combination with the detergent composition of the present invention. For example, hydrolase, oxidase, reductase, transferase, lyase, isomerase, ligase, synthetase and the like. Among them, proteases other than the present invention, cellulases, keratinases, esterases, cutinases, amylases, lipases, pullulanases, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases and the like are preferred, and proteases, cellulases, amylases and lipases are particularly preferred. preferable. Commercially available proteases include Alcalase (registered trademark; Novozymes), Esperase (registered trademark; Novozymes), Savinase (registered trademark; Novozymes), Everase (registered trademark; Novozymes), cannase (registered trademark; Novozymes), Properase (registered trademark; Genencor Corporation), Pragfect (registered trademark; Genencor Corporation), KAP (Kao) and the like. Examples of cellulase include cellzyme (registered trademark; Novozymes), carezyme (registered trademark; Novozymes), KAC, alkaline cellulase produced by Bacillus sp. KSM-S237 described in JP-A-10-313859, Japanese Patent Application 2002 Mutated alkaline cellulase described in No. 116553 (Kao). Examples of the amylase include Termamyl (registered trademark; Novozymes), Duramil (registered trademark; Novozymes), Plaster (registered trademark; Genencor), KAM (Kao) and the like. Examples of the lipase include Lipolase (registered trademark; Novozymes), Lipolase Ultra (registered trademark; Novozymes), and the like.
[0038]
When a protease other than the alkaline protease of the present invention is used in combination in the detergent composition, the amount is preferably from 0.1 to 500 PU per kg of the detergent composition. When cellulase is used in combination, the amount is preferably 300 to 3,000,000 KU per kg of the detergent composition based on the unit (U) determined by the enzyme activity measurement method described in paragraph [0020] of JP-A-10-313859. When amylase is used in combination, the amount is preferably 50 to 500,000 IU per kg of the detergent composition based on the unit (IU) determined by the amylase activity measuring method described in paragraph [0040] of JP-A-11-43690. Further, when lipase is used in combination, it is preferable to use 10,000 to 1,000,000 LU per 1 kg of the detergent composition based on the unit (LU) determined by the lipase activity measurement method described in Example 1 of JP-T-8-500013.
[0039]
Known detergent components can be blended in the detergent composition of the present invention. Examples of the known detergent components include the following.
(1) Surfactant
The surfactant is incorporated in the detergent composition in an amount of 0.5 to 60% by mass, particularly 10 to 45% by mass for a powdery detergent composition, and 20 to 50% by mass for a liquid detergent composition. preferable. When the detergent composition of the present invention is a bleach or a detergent for automatic dishwashers, the surfactant is generally blended in an amount of 1 to 10% by mass, preferably 1 to 5% by mass.
Examples of the surfactant used in the cleaning composition of the present invention include one or a combination of an anionic surfactant, a nonionic surfactant, an amphoteric surfactant, and a cationic surfactant. However, preferred are anionic surfactants and nonionic surfactants.
Examples of the anionic surfactant include sulfates of alcohols having 10 to 18 carbon atoms, sulfates of alkoxylated alcohols having 8 to 20 carbon atoms, alkylbenzene sulfonates, paraffin sulfonates, α-olefin sulfones. Acid salts, α-sulfofatty acid salts, α-sulfofatty acid alkyl ester salts or fatty acid salts are preferred. In the present invention, in particular, a linear alkylbenzene sulfonate having 10 to 14 carbon atoms, more preferably 12 to 14 carbon atoms in the alkyl chain is preferable, and as a counter ion, an alkali metal salt or an amine is preferable. Alternatively, potassium, monoethanolamine, and diethanolamine are preferred.
Examples of the nonionic surfactant include polyoxyalkylene alkyl (8 to 20 carbon atoms) ether, alkyl polyglycoside, polyoxyalkylene alkyl (8 to 20 carbon atoms) phenyl ether, and polyoxyalkylene sorbitan fatty acid (8 to 20 carbon atoms). 22) Esters, polyoxyalkylene glycol fatty acid (8 to 22 carbon atoms) esters, and polyoxyethylene polyoxypropylene block polymers are preferred. In particular, as a nonionic surfactant, 4 to 20 mol of an alkylene oxide such as ethylene oxide or propylene oxide is added to an alcohol having 10 to 18 carbon atoms [having an HLB value (calculated by the Griffin method) of 10.5 to 15. 0, preferably 11.0 to 14.5].
[0040]
(2) Divalent metal ion scavenger
The divalent metal ion scavenger is incorporated in an amount of 0.01 to 50% by mass, preferably 5 to 40% by mass. Examples of the divalent metal ion scavenger used in the cleaning composition of the present invention include condensed phosphates such as tripolyphosphate, pyrophosphate and orthophosphate, aluminosilicates such as zeolite, and synthetic layered crystalline silicic acid. Salt, nitrilotriacetate, ethylenediaminetetraacetate, citrate, isocitrate, polyacetal carboxylate and the like. Among them, crystalline aluminosilicates (synthetic zeolites) are particularly preferable, and among A-type, X-type and P-type zeolites, A-type is particularly preferable. As the synthetic zeolite, those having an average primary particle size of 0.1 to 10 μm, particularly 0.1 to 5 μm are suitably used.
[0041]
(3) Alkali agent
The alkali agent is added in an amount of 0.01 to 80% by mass, preferably 1 to 40% by mass. In the case of a powder detergent, alkali metal carbonates such as sodium carbonate, which are collectively referred to as dense ash and light ash, and amorphous alkali metal silicates such as JIS Nos. 1, 2, and 3 are exemplified. These inorganic alkaline agents are effective in forming the skeleton of the particles when the detergent is dried, and a relatively hard detergent having excellent fluidity can be obtained. Other alkalis include sodium sesquicarbonate and sodium hydrogen carbonate, and phosphates such as tripolyphosphate also act as alkali agents. In addition, as the alkaline agent used in the liquid detergent, sodium hydroxide, as well as the above alkaline agent, and mono, di, or triethanolamine can be used, and can also be used as a counter ion of the activator.
[0042]
(4) Recontamination inhibitor
The anti-redeposition agent is added in an amount of 0.001 to 10% by mass, preferably 1 to 5% by mass. Examples of the anti-redeposition agent used in the cleaning composition of the present invention include polyethylene glycol, carboxylic acid-based polymers, polyvinyl alcohol, and polyvinylpyrrolidone. Among them, the carboxylic acid-based polymer has a function of trapping metal ions and a function of dispersing solid particle stains from clothes into a washing bath, in addition to the ability to prevent re-contamination. The carboxylic acid-based polymer is a homopolymer or a copolymer of acrylic acid, methacrylic acid, itaconic acid, or the like, and the copolymer is preferably a copolymer of the above monomer and maleic acid, and has a molecular weight of thousands to 100,000. preferable. In addition to the carboxylic acid-based polymers, polymers such as polyglycidylates, cellulose derivatives such as carboxymethylcellulose, and aminocarboxylic acids-based polymers such as polyaspartic acid also have metal ion scavengers, dispersants and anti-soil re-contamination ability It is preferred.
[0043]
(5) Bleach
For example, bleaching agents such as hydrogen peroxide and percarbonate are preferably blended in an amount of 1 to 10% by mass. When a bleaching agent is used, a bleaching activator (activator) such as tetraacetylethylenediamine (TAED) or JP-A-6-316700 can be blended in an amount of 0.01 to 10% by mass.
[0044]
(6) Fluorescent agent
Examples of the fluorescent agent used in the cleaning composition of the present invention include a biphenyl type fluorescent agent (eg, Tinopearl CBS-X) and a stilbene type fluorescent agent (eg, a DM type fluorescent dye). Preferably, the fluorescent agent is added in an amount of 0.001 to 2% by mass.
[0045]
(7) Other components
The detergent composition of the present invention contains a builder, a softening agent, a reducing agent (such as a sulfite), a foam inhibitor (such as silicone), a fragrance, and other additives known in the field of clothing detergents. Can be.
[0046]
The detergent composition of the present invention can be produced according to a conventional method by combining the alkaline protease of the present invention obtained by the above method and the above-mentioned known cleaning components. The form of the detergent can be selected according to the application, and for example, can be liquid, powder, granule, paste, solid, or the like.
The detergent composition thus obtained can be used as a clothing detergent, a bleach, a detergent for cleaning hard surfaces, a drainpipe detergent, a denture detergent, a germicidal detergent for medical instruments and the like.
[0047]
【Example】
[Protease activity measurement method-casein method]
After incubating 1.0 mL of 50 mM borate buffer (pH 10.5) containing 1% (w / v) casein at 30 ° C. for 5 minutes, 0.1 mL of the enzyme solution is added, and the reaction is performed for 15 minutes. 2.0 mL of a reaction stop solution (0.11 M trichloroacetic acid-0.22 M sodium acetate-0.33 M acetic acid) was added, and the mixture was allowed to stand at room temperature for 30 minutes, followed by filtration. Quantification was carried out by the modified method described above. That is, an alkaline copper solution (1% sodium potassium tartrate: 1% copper sulfate pentahydrate: 2% sodium carbonate, 0.1N sodium hydroxide = 1: 1: 100) is added to 0.5 mL of the filtrate. After 5 mL was added and left at room temperature for 10 minutes, 0.25 mL of a phenol solution [a phenol reagent (Kanto Chemical Co., Ltd.) diluted twice with distilled water] was added, and the mixture was kept at 30 ° C. for 30 minutes. Thereafter, the absorbance at 660 nm was measured. One unit of protease (1 PU) was defined as the amount of enzyme required to release an acid-soluble protein hydrolyzate corresponding to 1 mmol of tyrosine per minute under the above reaction conditions.
[0048]
Example 1
Random mutation was given to a range of about 2.0 kb including the stop codon of the alkaline protease structural gene derived from Bacillus sp. KSM-KP43 strain. As a random mutagenesis method, a Taq polymerase having no error repairing ability: Takara Taq (Takara) was used as a DNA polymerase in order to utilize a base incorporation error during PCR. First, PCR was performed using primer 1 (5'-AAATGGATCCGTGAGGGAGGGAACCGAATGAGAAAGAAGAAAAAGGTGTG-3 ', SEQ ID NO: 2) and primer 2 (5'-ATATTCTAGACGATTTACCATTAATTCCTCTCACCC-3', SEQ ID NO: 3) capable of amplifying the above-described DNA of about 2.0 kb. Primer 1 is at the 5 'end of the sense strand Bam An HI linker was added to Primer 2 at the 5 'end of the antisense strand. Xba An I linker was provided. The reaction system was a 100 μL system containing 10 ng of template DNA, 10 pmol of each primer, 20 nmol of each dNTP, 10 μL of a reaction buffer attached to Takara Taq, and 2.5 U of Taq polymerase. The PCR conditions were as follows: denaturation of the template DNA at 94 ° C. for 2 minutes, followed by 30 cycles of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes. The PCR product was purified using a PCR product purification kit (Roche) and eluted with 100 μL of sterile water. Next, a second PCR was performed using 1 μL of the eluate as a template DNA, and the obtained PCR product was purified and used for subsequent experiments.
[0049]
A terminal restriction enzyme linker of the amplified DNA fragment of about 2.0 kb was Bam HI, Xba Cut with I (Roche). As an expression vector into which the amplified DNA is to be integrated, pHA64 (Japanese Patent Application No. 8-332050: downstream of the promoter 64) capable of replicating in Bacillus bacteria. Bam HI, Xba (With an I site). Bam HI, Xba I-treated amplified DNA fragment and Bam HI, Xba After mixing the pHA64 treated with I, a ligase reaction was performed by Ligation High (Toyobo). DNA was recovered from the ligase reaction solution by ethanol precipitation and used as DNA for subsequent transformation.
[0050]
Bacillus sp. KSM-KP43 (hereinafter abbreviated as KP-43 strain) was used as a host to be transformed. The transformation was performed by electroporation, and the transformation was performed using SSH-10 (Shimadzu Corporation) and Gene Pulser Cuvette (Bio-Rad).
[0051]
The transformant of the KP43 strain was transformed into an alkaline agar medium containing skim milk [Skim milk (Difco) 1% (w / v), bactotripton (Difco) 1%, yeast extract (Difco) 0.5%, sodium chloride 0.5% , Agar 1.5%, anhydrous sodium carbonate 0.05%, tetracycline 15 ppm], and the presence or absence of the protease gene was determined based on the halo formation status.
The transformed KP43 strain carrying the plasmid in which the protease gene was inserted into pHA64 was selected and used for subsequent culture.
[0052]
For each transformant, single colonies were separated and halo formation was confirmed. In a test tube, a 5 mL seed culture medium [Polypeptone S (Nippon Pharmaceutical) 6.0% (w / v), yeast extract 0.1%, maltose 1 0.0%, magnesium sulfate heptahydrate 0.02%, potassium dihydrogen phosphate 0.1%, anhydrous sodium carbonate 0.3%, tetracycline 30 ppm], and pre-cultured at 30 ° C. and 320 rpm overnight. did. This seed culture was added to a 20 mL main medium [polypeptone S 8% (w / v), yeast extract 0.3%, maltose 10%, magnesium sulfate heptahydrate 0.04%, phosphoric acid 2 in a 500 mL Sakaguchi flask. 1% was inoculated (v / v) into 0.2% of potassium hydrogen, 1.5% of anhydrous sodium carbonate, and 30 ppm of tetracycline], and cultured at 30 ° C. and 121 rpm for 3 days. The obtained culture was centrifuged, and the protease activity in the culture supernatant was measured. The protease activity was measured by the casein method, and the protein amount was measured using a protein assay kit (Wako Pure Chemical Industries, Ltd.). By comparing the value of the culture supernatant obtained when the transformant having the wild-type enzyme gene was cultured under the same conditions, a mutant protease gene in which the protease activity was improved was selected. Since the amount of protein in the culture supernatant increased almost in proportion to the protease activity, it was suggested that the obtained mutant had introduced a mutation necessary for improving the amount of secreted protein. Was done.
[0053]
Plasmids were recovered from the selected transformants using High pure plasmid isolation kit (Roche), and the nucleotide sequence was determined. Using 300 ng of plasmid DNA as a template, PCR was performed in a 20 μL reaction system using primers and Big Dye DNA Sequencing kit (Applied Biosystem), and subjected to analysis using DNA Sequencer 377 (Applied Biosystem).
[0054]
As a result, the mutants having improved protease activity showed that threonine at position 65 was proline, glycine at position 101 was asparagine, valine at position 273 was isoleucine, tyrosine at position 320 was phenylalanine, threonine at position 359 was serine, and serine at position 387 was serine. Was replaced with alanine, respectively, which resulted in about 5% improvement in protease activity (Table 2).
[0055]
Furthermore, the improvement of protease activity was examined by combining the above mutation sites. As a means of site-specific mutation, Site-Directed Mutagenesis System Mutan-Super Express Km kit was used, and combinations of mutation sites were examined using the following primers. Primer 3: Substitute threonine (T) at position 65 with proline (P) (5′-AATGCCAATGATCCGAATGGTCCATG-3 ′, SEQ ID NO: 4)
Primer 4: Substitute glycine (G) at position 101 with asparagine (N) (5′-TCTACATGGGATAGCAATGGGGGACTTGGAGG-3 ′, SEQ ID NO: 5) Primer 5: Substitute valine (V) at position 273 with isoleucine (I) ( 5'-CTTCGTGAGCATTTTATCAAAAACAGAGGCATC-3 ', SEQ ID NO: 6) Primer 6: Substituting tyrosine (Y) at position 320 with phenylalanine (F) (5'-AACGTTGCCTTTTGTGAACGAGTCC-3', SEQ ID NO: 7)
Primer 7: Substitute threonine (T) at position 359 with serine (S) (5′-GCGAGCACCATCTGCTTCCGTAACG-3 ′, SEQ ID NO: 8)
Primer 8: Substituting serine (S) at position 387 with alanine (A) (5′-TGACTTTACTGCGCCATACAATGATAAC-3 ′, SEQ ID NO: 9)
[0056]
The preparation of the template plasmid for mutagenesis was carried out in the multiple cloning site of pKF18k having an amber mutation marker for kanamycin selection. Bam HI, Xba It was constructed by introducing the mutant protease gene obtained in the above screening into the I site.
[0057]
Takara LA Taq (Takara) was used for PCR for site-specific mutagenesis. Mutagenesis PCR was performed using 5 pmol of each of the selection primers (attached to the Mutan-Super Express Km kit) and the primers 3 to 8 for mutagenesis, in which the 5 'end was phosphorylated, and 10 ng of the template plasmid. The reaction conditions were as follows: denaturation of the template DNA at 94 ° C. for 2 minutes, and 30 cycles of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 4 minutes. A mutant plasmid was obtained by transforming Escherichia coli MV1184 strain using the obtained PCR product. The mutation site of the obtained mutant plasmid was confirmed according to the above-described nucleotide sequencing method.
[0058]
By introducing the protease gene mutated by the site-specific mutation into pHA64, transforming the KP-43 strain, and culturing it under the conditions described above, a combination of mutated sites that further increase the protease activity as compared to the wild-type enzyme It was investigated.
[0059]
As a result, T65P + S387A, T359S + S387A, T65P + V273I + Y320F, T65P + V273I + T359S, T65P + V337A + S387A, T65P + Y320F + S387A, and T65P + G101N + S3A in the combination of the T30P + V3S1A and T65P + V273I were found in the T + T3 + 35A in the combination of the T + T3A + 30% were found in the T + T3A + 30% of the T + P + V273I.
[0060]
Further, using the following primers, the amino acids at positions 65, 101, 273, 320, 359, and 387 are substituted with any amino acids, and the amino acid at each position is replaced with another amino acid other than the above-mentioned substituted amino acid. We examined whether substitution is possible, and further examined the combination.
Primer 9: Substituting threonine (T) at position 65 with any amino acid (X) (5′-CGAATAATGCCAATGATNNNAATGGTCCATGGTACGC-3 ′, SEQ ID NO: 10)
Primer 10: Substituting glycine (G) at position 101 with an arbitrary amino acid (X) (5′-CTATCATGGGATAGCNNNGGGGGACTTGGAGG-3 ′, SEQ ID NO: 11)
Primer 11: Replace valine (V) at position 273 with any amino acid (X) (5′-CGTGGACATTTTNNNNAAAACAGAGGCCATCACACC-3 ′, SEQ ID NO: 12)
Primer 12: Substituting tyrosine (Y) at position 320 with an arbitrary amino acid (X) (5′-CCCTGAACGTTGCCNNNNGTGAACGAGTCC-3 ′, SEQ ID NO: 13)
Primer 13: Substituting threonine (T) at position 359 with an arbitrary amino acid (X) (5′-CCCCTGCGAGCACANNNGCTTCCGTAACGC-3 ′, SEQ ID NO: 14)
Primer 14: Substituting serine (S) at position 387 with any amino acid (X) (5′-GGAATGACTTTACTNNNCCATACAATGATAACTGG-3 ′, SEQ ID NO: 15)
[0061]
As a result, substitution of valine at position 273 by glycine or threonine other than isoleucine, substitution of tyrosine at position 320 by valine, threonine, leucine, isoleucine, or glycine other than phenylalanine, and threonine at position 359 by leucine or valine other than serine , Isoleucine, glutamine, and serine at position 387 were replaced with lysine, glutamine, glutamic acid, arginine, and histidine in addition to alanine. It was confirmed that the above amino acid substitution was possible.
[0062]
The combination Roh part Roh result wo shown door, T65P + Y320F + S387E, T65P + Y320G + S387A, T65P + V273I + T359S + S387E, T65P + V273I + T359L + S387A, T65P + V273I + T359I + S387A, T65P + V273G + T359S + S387A, T65P + V273I + T359S + S387K, T65P + V273I + T359V + S387A, T65P + V273I + T359Q + S387A, T65P + V273I + Y320T + S387A, T65P + V273I + Y320F + S387E, T65P + V273I + Y320F + S387K, T65P + V273T + Y320F + S387A, T65P + V273I + Y320L + S387H, T65P + 273I + Y320V + S387Q, improvement in T65P + V273I + Y320I + S387Q 10~30% in combination with the protease activity was observed (Table 2).
[0063]
[Table 2]

[0064]
Alkaline protease obtained by the combination of the above mutation sites, except for enhancing the secretion of the enzyme in the transformant, has the properties of the alkali-protease, that is, has an oxidizing agent resistance, and inhibits the casein degrading activity by a high concentration of fatty acids. , And the molecular weight observed by SDS-PAGE was 43,000 ± 2,000, confirming that the compound had the property of being active in the alkaline region.
Example 2
(1) Preparation of detergent
1m with stirring blade ³ 465 kg of water was added to the mixing tank, and after the water temperature reached 55 ° C., 135 kg of a 40% (w / v) aqueous sodium polyacrylate solution was added. After stirring for 15 minutes, 120 kg of sodium carbonate, 60 kg of sodium sulfate, 9 kg of sodium sulfite and 3 kg of a fluorescent dye were added. After further stirring for 15 minutes, 300 kg of zeolite was added and stirred for 30 minutes to obtain a homogeneous slurry (water content in the slurry was 50% by mass). This slurry was sprayed from a pressure spray nozzle installed near the top of the spray-drying tower to obtain base granules (the high-temperature gas supplied to the spray-drying tower was supplied at a temperature of 225 ° C. from the lower part of the tower, and from the top of the tower. Discharged at 105 ° C). Next, 100 parts by mass of the base granules are charged into a Lodige mixer (manufactured by Matsusaka Giken Co., Ltd., capacity 20 L, with jacket), and under stirring of the main shaft (150 rpm), 20 parts by mass of a nonionic surfactant, A mixture of 22 parts by mass of sodium alkyl (10 to 13 carbon atoms) benzenesulfonate, 4 parts by mass of sodium fatty acid (14 to 18 carbon atoms), 2 parts by mass of polyethylene glycol, and 4 parts by mass of water is charged for 3 minutes, and then Stirring was performed for 5 minutes. Further, 20 parts by mass of crystalline sodium silicate and 10 parts by mass of zeolite were charged into this mixer, and the surface was coated to obtain a detergent base.
The final granular detergent A was obtained by mixing 0.5% by mass of the protease particles of the present invention and 0.5% by mass of a fragrance to 99% by mass of the detergent base.
[0065]
(2) Raw materials used
Nonionic surfactant: Emulgen 108KM having an average ethylene oxide mole number of 8.5 (manufactured by Kao Corporation)
Aqueous sodium polyacrylate solution: Average molecular weight 10,000 (produced according to the method described in Examples of Japanese Patent Publication No. 2-24283)
Sodium carbonate: Dense ash (Central Glass Co., Ltd.)
Zeolite: zeolite type 4A having an average particle size of 3.5 μm (manufactured by Tosoh Corporation)
Polyethylene glycol: K-PEG6000 (average molecular weight 8500, manufactured by Kao Corporation)
Crystalline sodium silicate: Powder SKS-6 (manufactured by Hoechst Tokuyama Corporation)
Protease particles of the present invention: Granules (6 PU / g) prepared from each purified preparation of the alkaline protease of the present invention shown in Table 2 based on the method described in Example 1 of JP-A-62-257990.
Fluorescent dye: Tinopearl CBS-X (manufactured by Ciba Geigy)
[0066]
Example 3
(1) Preparation of detergent
First, a slurry having a solid content of 50% by mass was spray-dried at a hot air temperature of 250 ° C., and 7% by mass of sodium polyacrylate (mass average molecular weight 10,000), 26% by mass of sodium carbonate, 20% by mass of sodium sulfate, 6% by mass of sodium chloride, Base granules of 0.5% by mass of fluorescent dye, 40% by mass of zeolite and 0.5% by mass of water were obtained.
Next, 100 parts by mass of the above-mentioned base granules were put into a Lodige mixer (manufactured by Matsusaka Giken Co., Ltd., capacity 20 L, with jacket), and under stirring of the main shaft (150 rpm), 20 parts by mass of a nonionic surfactant, A mixture of 22 parts by mass of sodium alkyl (10 to 13 carbon atoms) benzenesulfonate, 4 parts by mass of sodium fatty acid (14 to 18 carbon atoms), 2 parts by mass of polyethylene glycol, and 4 parts by mass of water is added for 3 minutes, and then Stirring was performed for 5 minutes. Further, 20 parts by mass of crystalline sodium silicate and 10 parts by mass of zeolite were added to this mixer, and the surface was coated to obtain a detergent base.
The final granular detergent B was prepared by mixing 2.8% by mass of bleach particles, 1.2% by mass of bleach activator particles, 0.5% by mass of the protease particles of the present invention, and 0.5% by mass of fragrance to 95% by mass of the detergent base. Got.
[0067]
(2) Raw materials used
Nonionic surfactant: Emulgen 108KM having an average ethylene oxide mole number of 8.5 (manufactured by Kao Corporation)
Aqueous sodium polyacrylate solution: Average molecular weight 10,000 (produced according to the method described in Examples of Japanese Patent Publication No. 2-24283)
Sodium carbonate: Dense ash (Central Glass Co., Ltd.)
Zeolite: zeolite type 4A having an average particle size of 3.5 μm (manufactured by Tosoh Corporation)
Polyethylene glycol: K-PEG6000 (average molecular weight 8500, manufactured by Kao Corporation)
Crystalline sodium silicate: SKS-6 (manufactured by Hoechst Tokuyama Corporation)
Protease particles of the present invention: Granules (6 PU / g) prepared from each purified preparation of the alkaline protease of the present invention shown in Table 2 based on the method described in Example 1 of JP-A-62-257990.
Fluorescent dye: Tinopearl CBS-X (manufactured by Ciba Geigy)
Bleach particles: Sodium carbonate / hydrogen peroxide adduct (obtained in the same manner as bleach particles described in paragraph [0019] of JP-A-2000-256699)
Bleaching activator particles: Granulated sodium lauroyloxybenzenesulfonate (obtained in the same manner as bleach activator particles described in paragraph [0018] of JP-A-2000-256699).
[0068]
Example 4
Liquid detergent compositions (detergent C and detergent D) shown in Table 3 were prepared.
[0069]
[Table 3]

[0070]
1) Polyoxyethylene (average addition of 7 mol) alkyl ether having an alkyl group derived from a secondary alcohol having 12 to 14 carbon atoms (sophthanol 70, manufactured by Nippon Shokubai Chemical Industry)
2) Polyoxyethylene (average addition of 12 mol) alkyl ether having an alkyl group derived from a secondary alcohol having 12 to 14 carbon atoms (sophthanol 120, manufactured by Nippon Shokubai Chemical Industry)
3) A linear primary alcohol having 10 to 14 carbon atoms in which EO is added in an average of 5 mol, PO is added in an average of 2 mol, and EO is added in an average of 3 mol in the order of blocks.
4) Polyoxyethylene lauryl ether and EO added on average 8 mol 5) Polyoxyethylene lauryl ether and EO added on average 11.5 mol
6) Narrow range polyoxyethylene alkyl (sec-C ₁₂ / C ₁₃ )ether
7) Sodium linear alkylbenzene sulfonate having 10 to 14 carbon atoms
8) Amide / ether-modified silicone polymer (BY16-906, manufactured by Dow Corning Toray Silicone Co., Ltd.)
9) Phenoxypolyethylene glycol, acrylic acid, maleic acid copolymer synthesized by the method described on page 11, lines 6 to 13 of JP-A-10-60476 (mass average molecular weight 10,000, solid content 51.2%)
10) Penten / maleic acid (50/50 molar ratio) copolymer sodium salt (mass average molecular weight 7000)
11) Each purified sample of the alkaline protease of the present invention described in Table 2 (15 PU / g)
[0071]
Example 5
Among the compositions shown in Table 4 below, while stirring and mixing sodium percarbonate and sodium carbonate (dense ash), a 40% aqueous solution of sodium polyacrylate and sodium linear alkylbenzene sulfonate, or a nonionic surfactant, Alternatively, sodium lauroyloxybenzenesulfonate was added. Subsequently, the protease particles of the present invention prepared according to the method described in Example 1 of JP-A-62-257990 were added, and the mixture was stirred to a degree that the whole was uniform to prepare a bleaching agent.
[0072]
[Table 4]

[0073]
1) 500-700 μm particle size
2) Sodium linear alkylbenzene sulfonate having 12 to 14 carbon atoms
3) Polyoxyethylene alkyl ether (alkyl group having 12 to 14 carbon atoms, EO average addition mole number of 12)
4) Average molecular weight 8,000
5) Granules (6 PU / g) prepared from each purified preparation of the alkaline protease of the present invention described in Table 2 based on the method described in Example 1 of JP-A-62-257990.
[0074]
Example 6
The detergent compositions for fully automatic dishwashers shown in Table 5 below (detergents G and H) were prepared.
[0075]
[Table 5]

[0076]
1) Polyoxyethylene-polyoxypropylene copolymer (average molecular weight 2,000)
2) adduct of 7 mol of ethylene oxide and 8.5 mol of propylene oxide of a sec-alcohol having 12 to 14 carbon atoms
3) JIS No. 2 sodium silicate
4) Acrylic acid-maleic acid copolymer
5) Duramill 60T ^(TM) (Manufactured by Novozymes)
6) Granules (6 PU / g) prepared from each purified preparation of the alkaline protease of the present invention shown in Table 2 based on the method described in Example 1 of JP-A-62-257990.
[0077]
Example 7
Using the components shown in Table 6 below, a cleaning composition for hard surfaces (detergent J) was obtained.
[0078]
[Table 6]

[0079]
1) Sodium polyoxyethylene (EOP = 4) alkyl (C12) ether sulfate
2) Polyoxyethylene (EOP = 8) alkyl (C12) ether
3) alkyl (C12) polyglucoside (condensation degree 1.3)
4) Mono long chain tertiary alkyl (C12) dimethylamine oxide
5) Alkyl (C12) hydroxydimethylsulfobetaine
6) Molecular weight 10,000
7) Each purified sample of the alkaline protease of the present invention described in Table 2 (15 PU / mL)
[0080]
Example 8
Using the above detergent A (see Example 2), granular detergents shown in Table 7 below were obtained.
[0081]
[Table 7]

[0082]
1) Granules (6 PU / g) prepared from each purified preparation of the alkaline protease of the present invention described in Table 2 based on the method described in Example 1 of JP-A-62-257990.
2) Protease K-16 described in JP-A-5-25492 was adjusted to 5 PU / g based on the method described in Example 1 of JP-A-62-257990.
3) KAC-500 (manufactured by Kao Corporation)
4) Lipolase 100T ^(TM) (Manufactured by Novozymes)
[0083]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, while having activity even in the presence of a high concentration of fatty acids, it is possible to provide an alkaline protease having a high secretion ability, as well as having excellent detergency against complex stains including not only proteins but also sebum. .
[0084]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram in which the amino acid sequences of alkaline proteases having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 are aligned.

Claims (7)

The amino acid residue at (a) position 65, (b) position 101, (c) position 273, (d) position 320, (e) position 359, (f) position 387 or a position corresponding thereto in SEQ ID NO: 1 The following amino acid residues;
(A) position: proline,
(B) location: asparagine,
(C) Position: isoleucine, glycine, threonine,
(D) position: phenylalanine, valine, threonine, leucine, isoleucine, glycine,
(E) Position: serine, leucine, valine, isoleucine, glutamine,
(F) Position: alanine, lysine, glutamine, glutamic acid, arginine, histidine,
Alkaline protease selected from the group consisting of: In an alkaline protease having an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 80% or more homology with the amino acid sequence, (a) position 65, (b) position 101, (c) position 273, (d) ) The amino acid residue at position 320, (e) position 359, (f) position 387 or a position corresponding thereto is the following amino acid residue;
(A) position: proline,
(B) location: asparagine,
(C) Position: isoleucine, glycine, threonine,
(D) position: phenylalanine, valine, threonine, leucine, isoleucine, glycine,
(E) Position: serine, leucine, valine, isoleucine, glutamine,
(F) Position: alanine, lysine, glutamine, glutamic acid, arginine, histidine,
Alkaline protease selected from the group consisting of: A gene encoding the alkaline protease according to claim 1. A recombinant vector containing the gene according to claim 3. A transformant containing the vector according to claim 4. The transformant according to claim 5, wherein the host is a microorganism. A detergent composition containing the alkaline protease according to claim 1.

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