JP2018050618A - 変異体微生物のプロテアーゼを含む組成物及び方法 - Google Patents
変異体微生物のプロテアーゼを含む組成物及び方法 Download PDFInfo
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Abstract
【解決手段】少なくとも一つの修飾を持つスブチリシン変異体を提供し、少なくとも一つの修飾はG100C、S101D、S101H、Q103D、M124G、L126G、S132H、S161N、Q275Zから選択され及び/又は以下のもの等から選択される:A001T/L096V/G097S/A098D/D099I、A001V/G097S/A098R/D099G、等。
【選択図】図2A
Description
ヘミセルラーゼ、ペルオキシダーゼ、プロテアーゼ、セルラーゼ、キシラナーゼ、リパーゼ、フォスフォリパーゼ、エステラーゼ、クチナーゼ、ペクチナーゼ、ケラチナーゼ、レダクターゼ、オキシダーゼ、フェノールオキシダーゼ、リポオキシダーゼ、リグリナーゼ、プルラナーゼ、タンナーゼ、ペントサナーゼ、マラナーゼ、β‐グルカナーゼ、アラビノシダーゼ、ヒアルニダーゼ、コンドロイチナーゼ、ラッカーゼ、及びアミラーゼ又はこれらの混合物である。さらなる実施の態様においては、洗浄組成物はさらに少なくとも一つの安定剤を含む。さらなる実施の態様においては、洗浄組成物は本明細書に記載の少なくとも一つの任意のスブチリシン変異体を少なくとも0.0001重量%含み、そして任意選択的に少なくとも一つの好適な付加成分を含む。
本発明は本明細書に記載の変異体プロテアーゼ及び少なくとも一つの変異体スブチリシンを含む組成物を提供する。ある実施の態様においては、本発明は、ポリペプチド分解又は合成が求められる以下の種々の商業的分野の応用においてプロテアーゼを生産する方法を提供する:すなわち、洗浄組成物を含み、また、飼料用成分、繊維加工、皮革仕上げ、穀物処理、肉加工、食料品加工、タンパク質加水分解物、消化補助剤、微生物構成物、静菌性組成物、静真菌性組成物及びパーソナルケア製品(例えば、口腔ケア、ヘアーケア、及びスキンケア組成物)がある。本発明はさらに、現在使用されているスブチリシンプロテアーゼと比べてそれに匹敵し又は改良された洗浄能力を持つ酵素組成物を提供する。
本発明の洗浄組成物は例えば、洗濯に使用された場合多くの利点を持つ。事実低温液で使用された場合効果が増大すると言うユニークな利点をもつため本発明の酵素は洗濯での応用に理想的に適する。さらに、本発明の酵素は顆粒状及び液体状の組成物で使用することができる。
その様な固体組成物のpHは、溶媒が蒸留水であるその組成物の10%固体溶液として測定される。これらの実施の態様においては、全てのpHは20℃で測定される。
この意味で、本発明の変異体プロテアーゼは技術分野で知られている任意のカプセル化材料によりカプセル化される。ある実施の態様においては、カプセル化材料は通常本発明の変異体プロテアーゼの触媒の少なくとも一部をカプセル化する。通常カプセル化材料は水溶性及び/又は水分散性である。ある実施の態様においては、カプセル化材料はガラス転移温度が0℃以上である。ガラス転移温度はWO 97/11151にさらに詳しく記載されている。カプセル化材料は炭水化物、天然又は合成ゴム、キチン、キトサン、セルロース及びセルロース誘導体、ケイ酸塩、リン酸塩、ホウ酸塩、ポリビニルアルコール、ポリエチレングリコール、パラフィンワックス、及びこれらの組み合わせからなる群れから選択される。カプセル化材料が炭水化物である場合、それは単糖類、オリゴ糖、多糖類及びこれらの組み合わせから選択される。通常カプセル化材料はデンプンである。好適なデンプンはEP 0 922 499; 米国特許第4,977,252号; 第5,354,559号;及び第5,935,826号に記載されている。ある実施の態様においては、カプセル化材料は熱可塑剤、アクリロニトリル、メタアクリロニトリル、ポリアクリロニトリル、ポリメタアクリロニトリル、及びこれらの混合物の様なプラスチックからなるミクロスフィアであり、使用可能な、市場で購入可能なミクロスフィアはEXPANCEL(登録商標) (Stockviksverken, Sweden), 及びPM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES(登録商標)、LUXSIL(登録商標)、Q-CEL(登録商標)、及びSPHERICEL(登録商標) (PQ Corp., Valley Forge, PA)の名称で提供されるものを含む。
ある実施の態様においては、セルラーゼは成熟野生型又は変異体セルラーゼの部分又は断片として組み入れられ、N末端の一部は欠失している(例えば、米国特許第5,874,276号を参照)。ある実施の態様においては、本発明の洗浄組成物はさらに組成物の重量の約0.00001 % から約10%のレベルのセルラーゼ及び残りの部分は組成物の重量で洗浄付加材料を含む。本発明の他の特徴としては、本発明の洗浄組成物はまた組成物の重量の約0.0001% から約10%, 約 0.001%から約5%, 約0.001%から約2%, 約0.005%から約0.5%のレベルのセルラーゼを含む。
例えば、米国特許第5,576,282号に開示されているマンガンベース触媒を含む。さらに、本発明で有用なコバルト漂白触媒が知られており、例えば、米国特許第5,597,936号及び第5,595,967号に開示されている。その様なコバルト触媒は米国特許第5,597,936号及び第5,595,967号の実施例により教示されるように、知られた手順により容易に生成することができる。ある実施の態様においては、本明細書に記載の組成物はまた巨大多環式剛性リガンド(すなわち、「MRL」)を好適に含む。実際問題として、発明を限定する意味ではないが、本明細書に記載の組成物及び洗浄プロセスは調節が可能であり、水性洗濯媒体中で活性MRL種を少なくとも1ppmのレベルで提供し、好ましくは約0.005 ppmから約25 ppm、より好ましくは約0.05 ppmから約10 ppm、最も好ましくは約0.1ppm から約5 ppmの範囲のMRLを提供する。
特に断らない限り、本発明においてはタンパク質操作、分子生物学、微生物学、及び組み換えDNAで通常使用される従来の技術を用い、これらの技術は当業者の常識の範囲内にある。その様な技術は当業者に知られており、また当業者に知られている多くの書籍及び参考文献に記載されている(例えば、Sambrook et al.,「分子クローニング(実験室マニュアル)」("Molecular Cloning: A Laboratory Manual), Second Edition (Cold Spring Harbor), [1989]); 及びAusubel et al., 「分子生物学における現在のプロトコール」("Current Protocols in Molecular Biology" )[1987])。本明細書に記載の全ての特許、特許出願、論文及び刊行物は参照により本明細書に組み入れられる
本明細書において、別の意味を持つものとして規定しない限り、本明細書の全ての技術及び科学用語はこの発明が関係する技術分野の当業者により通常理解されると同じ意味を持つ。これらの用語の定義を提供する、当業者に知られた多くの辞書が利用可能である。本明細書に記載のこれらの方法と同様又は同等な方法及び材料は、本発明の実施に用いることができるが、本明細書では好ましい方法及び材料が記載されている。したがって、以下に続いて定義される用語は明細書を全体として考察することにより、より完全に把握することができる。
これらは当業者により使用される状況により変わりうるからである。
両文献は参照により本明細書に組み入れられる。)pNAアッセイ(例えば、Del Mar他、Anal Biochem, 99:316-320 [1979]を参照願いたい)はまた、勾配溶離中に回収される部分の活性酵素濃度を決定するのに用いることが出来る。このアッセイは、酵素が可溶合成基質、スクシニルーアラニンーアラニンープロリンーフェニルアラニンーp−ニトロアニリド(sAAPF-pNA)を加水分解するに連れてp−ニトロアニリンが放出される速度を測定する。加水分解反応から黄色の生成される速度が分光光度計において410nm波長で測定され、これは活性酵素濃度に比例する。さらに280nmでの吸光測定は全タンパク質濃度の決定に用いることができる。活性酵素/全タンパク質の比率は酵素純度を示す。
好適な発現ベクターを選択することは当業者の常識の範囲にある。「発現カセット」(expression casette)の用語は本明細書において「DNA構築体」及びその文法的同等体と相互交換的に用いられる。適当な発現ベクターを選択することは当業者の常識の範囲内にある。
ある実施の態様においては、関心の対象のタンパク質は細胞間で発現し、他の実施の態様においては、関心の対象のタンパク質は分泌ポリペプチドである。特に好ましい実施の態様においてはこれらの酵素は本明細書に記載のセリンプロテアーゼを含む。ある実施の態様においては、関心の対象のタンパク質はシグナルペプチドに融合された分泌ポリペプチドである(すなわち、分泌されるタンパク質上のアミノ末端伸張である)。殆んど全ての分泌タンパク質はアミノ末端タンパク質伸張を用い、それは細胞膜に渡り前駆体タンパク質を標的にし、且つ前駆体タンパク質の転座に決定的な役割を果す。この伸張は膜転写の間又はその直後にシグナルペプチダーゼによってタンパク質分解活性により除去される。
ここで言う「同等」(equivalent)の用語は配列番号1に示す配列を持つポリヌクレオチドに、中程度から最大に厳格な条件でハイブリダイズすることができるポリヌクレオチドによりコードされるセリンプロテアーゼ酵素を言う。例えば、同等であることは、同等の成熟セリンプロテアーゼが、配列番号2のアミノ酸配列を持つ成熟スブチリシンプロテアーゼと、少なくとも約 70%, 少なくとも約 75%, 少なくとも約 80%, 少なくとも約 85%, 少なくとも約 90%, 少なくとも約 91 %, 少なくとも約 92%, 少なくとも約 93%, 少なくとも約 94%, 少なくとも約 95%, 少なくとも約 96%, 少なくとも約 97%, 少なくとも約 98%及び/又は少なくとも約 99%の配列を持つことを意味する。
ある好ましい実施の態様においては、修飾された配列の発現生成物は短縮タンパク質(例えば、修飾が配列の欠失、又は中断である場合)である。ある特に好ましい実施の態様においては、短縮されたタンパク質は生物学的活性を維持する。他の実施の態様においては、修飾された配列の発現生成物は細長いタンパク質である(例えば、修飾は核酸配列中に挿入を含む)。ある実施の態様においては、挿入により短縮タンパク質となる(例えば、挿入がストップコドンを形成する場合)。この様に、挿入は、短縮タンパク質又は発現生成物として細長いタンパク質となることもある。
これらのスブチリシンプロテアーゼには以下を含むがこれに限定されるものではない:
OPTIMASE(登録商標)プロテアーゼ (Genencor), PURAFECT(登録商標)プロテアーゼ製品(Genencor), SAVINASE(登録商標)プロテアーゼ (Novozymes), BPN' −変異体(例えば、米国特許Re 34,606を参照), RELASE(登録商標), DURAZYME(登録商標)、EVERLASE(登録商標)、KANNASE(登録商標)プロテアーゼ (Novozymes), MAXACAL(登録商標), MAXAPEM(登録商標), PROPERASE(登録商標)プロテアーゼ (Genencor;また米国特許Re 34,606, 及び米国特許第5,700,676号; 第5,955,340号; 第6,312,936号;及び第6,482,628号を参照), 及びバチルス レンツス(Bacillus lentus)変異体プロテアーゼ製品 (例えば、WO 92/21760, WO 95/23221及び/又はWO 97/07770に記載のもの)。
本発明はさらに以下の実施例において詳細に説明する。これらの実施例は本願発明の範囲を限定するものと解してはならない。添付の図面は本明細書の不可分の一部であると理解されるべきである。以下の実施例は本願発明を説明するために記載されているもので、その発明の範囲を限定するものではない。
PI又はPi(効果指数)、ppm(百万分の一);M (モルの); mM (ミリモルの); μM (ミクロモルの);nM(ナノモルの);mol(モル); mmol(ミリモル);μmol (ミクロモル);nmol(ナノモル); gm(グラム);mg(ミリグラム);μg(ミクログラム);pg(ピコグラム);L(リッター);ml 及びmL(ミリリットル);μl及びυL(ミクロリットル);cm(センチメータ);mm (ミリメータ); μm (ミクロメータ); nm (ナノメータ); U (単位); V (ボルト); MW (分子量); sec (秒); min(s) (分); hr(s) (時間); ℃(摂氏度);QS (十分な量); ND (実施せず); NA (適用なし); rpm(分当り回転数);H2O(水);dH2O(非イオン水);HCL(塩酸);aa及びAA(アミノ酸);bp(塩基ペア);kb(キロ塩基ペア);kD(キロダルトン); cDNA (DNAのコピー又は相補DNA); DNA (デオキシリボ核酸); ssDNA (単鎖DNA); dsDNA (二重鎖DNA); dNTP (三リン酸デオキシリボ核酸); RNA (リボ核酸); MgCl2 (塩化マグネシウム); NaCl (塩化ナトリウム); w/v (重量対容積); v/v (容積体容積);g(重力);OD(光学濃度);Dulbeccoリン酸緩衝生理食塩水(DPBS); SOC((2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl); Terrific Broth (TB; 12 g/1 Bacto Tryptone, 24 g/1 グリセロール, 2.31 g/1 KH2PO4及び12.54 g/1 K2HPO4); OD280 (280 nmの光学濃度); OD600 (600 nmの光学濃度); A40S (405 nmの吸光度); Vmax(酵素触媒による初期最大速度);PAGE (ポリアクリルアミド ゲル電気泳動法); PBS (リン酸緩衝生理食塩水 [150 mM NaCl, 10 mM リン酸ナトリウム緩衝液, pH 7.2]); PBST (PBS+0.25% TWEEN(登録商標) 20); PEG(ポリエチレングリコール); PCR (ポリメラーゼ連鎖反応); RT-PCR (逆転写PCR); SDS (ドデシル硫酸ナトリウム); Tris (トリ(ヒドロメチル)アミノメタン); HEPES(N-[2-ヒドロキシエチル]ピペザジン-N-[2-エタンスルフォン酸]); HBS (HEPES 緩衝生理食塩水); Tris-HCl (トリ [ヒドロキシメチル] アミノメタンーヒドロクロライド); Tricine (N- [トリ-(ヒドロキシメチル)- メチル] -グリシン); CHES (2-(N-シクロ-ヘキシルアミノ)エタン-スルホン酸); TAPS (3-{ [トリ-(ヒドロキシメチル)-メチル]- アミノ}-プロパンスルホン酸); CAPS (3-(シクロ-ヘキシルアミノ)- プロパンスルホン酸; DMSO (ジメチル スルホキシド); DTT (1,4-ジチオ-DL-スレイトール); SA (シナピン酸(s,5-ジメトキシ-4-ヒドロキシ桂皮酸); TCA (トリクロロ酢酸); Glut及びGSH (還元グルタチオン); GSSG (酸化グルタチオン); TCEP (トリ[2-カルボキシエチル]ホスフィン); Ci (キュリー); mCi (ミリキュリー); μCi (ミクロキュリー); HPLC (高圧液体クロマトグラフィ‐); RP-HPLC (逆相高圧液体クロマトグラフィ‐); TLC (薄層クロマトグラフィ‐); MALDI-TOF (マトリックス支援レーザー脱離/イオン化―飛行時間)(time of flight); Ts (トシル基); Bn (ベンジル基); Ph (フェニル基); Ms (メシル基); Et(エチル基), Me (メチル基); Taq (サーマス・アクアチクスDNA ポリメラーゼ); Klenow (DNAポリメラーゼI 大(Klenow)断片); EGTA (エチレングリコール‐ビス(β-アミノエチル エーテル)、N, N, N', N'-テトラ酢酸); EDTA (エチレンジアミンテトラ酢酸); bla (β-ラクタマーゼ又は抗アミピシリン遺伝子);HDL (高密度液体);
MJ Research (MJ Research, Reno, NV); Baseclear (Baseclear BV, Inc., Leiden, the Netherlands); PerSeptive (PerSeptive Biosystems, Framingham, MA); Thermo Finnig an (ThermoFinnigan, San Jose, CA); Argo (Argo BioAnalytica, Morris Plains, NJ);Seitz EKS (SeitzSchenk Filtersystems GmbH, Bad Kreuznach, Germany); Pall (Pall Corp., East Hills, NY); Spectrum (Spectrum Laboratories, Dominguez Rancho, CA); Molecular Structure (Molecular Structure Corp., Woodlands, TX); Accelrys (Accelrys, Inc., San Diego, CA); Chemical Computing (Chemical Computing Corp., Montreal, Canada); New Brunswick (New Brunswick Scientific, Co., Edison, NJ); CFT (Center for Test Materials, Vlaardingen, the Netherlands); Test Fabrics (Test Fabrics, Inc., West Pittiston, PA), Procter & Gamble (Procter & Gamble, Inc., Cincinnati, OH); Epicentre (Epicentre Biotechnologies, Madison, WI); GE Healthcare (GE Healthcare, Chalfont St. Giles, United Kingdom); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme (MegazymeInternational Ireland Ltd., Bray Business Park, Bray, Co., Wicklow, Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CP Kelco, Wilmington, DE); Corning (Corning Life Sciences, Corning, NY); (NEN (NEN Life Science Products, Boston, MA); Pharma AS (Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis, Lewisville, TX); ATCC (American Type Culture Collection, Rockville, MD); Gibco/BRL (Gibco/BRL, Grand Island , NY); Sigma (Sigma Chemical Co., St. Louis, MO); Pharmacia (Pharmacia Biotech, Piscataway, NJ); NCBI (National Center for Biotechnology Information); Applied Biosystems (Applied Biosystems, Foster City, CA); BD Biosciences and/or Clontech (BD Biosciences CLONTECH Laboratories, Palo Alto, CA); Operon Technologies (Operon Technologies, Inc., Alameda, CA); MWG Biotech (MWG Biotech, High Point, NC); Oligos Etc (Oligos Etc. Inc, Wilsonville, OR); Bachem (Bachem Bioscience, Inc., King of Prussia, PA); Difco (Difco Laboratories, Detroit, MI); Mediatech (Mediatech, Herndon, VA; Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); Oxoid (Oxoid Inc., Ogdensburg, NY); Worthington (Worthington Biochemical Corp., Freehold, NJ); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, MD); Millipore (Millipore, Billerica, MA); Bio-Rad (Bio-Rad, Hercules, CA); Invitrogen (Invitrogen Corp., San Diego, CA); New England Biolabs and NEB (New England Biolabs, Ipswich, MA); Sigma (Sigma Chemical Co., St. Louis, MO); Pierce (Pierce Biotechnology, Rockford, IL); Takara (Takara Bio Inc. Otsu, Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science (EM Science, Gibbstown, NJ); Qiagen (Qiagen, Inc., Valencia, CA); Biodesign (Biodesign Intl., Saco, Maine); Aptagen (Aptagen, Inc., Herndon, VA); Sorvall (Sorvall brand, from Kendro Laboratory Products, Asheville, NC); United States Testing (United States Testing Co., Hoboken, NJ);Molecular Devices (Molecular Devices, Corp., Sunnyvale, CA); R&D Systems (R&D Systems, Minneapolis, MN); Stratagene (Stratagene Cloning Systems, La Jolla, CA); Marsh (Marsh Biosciences, Rochester, NY); Geneart (Geneart GmbH, Regensburg, Germany); DNA2.0 (DNA2.0, Menlo Park, CA); Gene Oracle (Gene Oracle, Mountain View, CA); Zymo Research (Zymo Research, Orange, CA); Bio-Tek (Bio-Tek Instruments, Winooski, VT); Biacore (Biacore, Inc., Piscataway, NJ); PeproTech (PeproTech, Rocky Hill, NJ);SynPep (SynPep, Dublin, CA); New Objective (New Objective brand; Scientific Instrument Services, Inc., Ringoes, NJ); Waters (Waters, Inc., Milford, MA); Matrix Science (Matrix Science, Boston, MA); Dionex (Dionex, Corp., Sunnyvale, CA); Monsanto (Monsanto Co., St. Louis, MO); Wintershall (Wintershall AG, Kassel, Germany); BASF (BASF Co., Florham Park, NJ); Huntsman (Huntsman Petrochemical Corp., Salt Lake City, UT); Enichem (Enichem Iberica, Barcelona, Spain); Fluka Chemie AG (Fluka Chemie AG, Buchs, Switzerland); Gist-Brocades (Gist-Brocades, NV, Delft, the Netherlands); Dow Corning (Dow Corning Corp., Midland, MI); and Microsoft (Microsoft, Inc., Redmond, WA)。
アッセイ
以下の実施例において、理解を容易にするために種々のアッセイが実施された。規定のプロトコールからの逸脱については各実施例で示す。本発明ではスブチリシンの番号は以下に示す様に成熟BPN’配列の番号に対応する。
BPN’(例えば、参考プロテアーゼ)及びBPN’変異体について、このアッセイはマイクロタイタープレートにおいて、33℃で、230 rpmで振動させそして湿気のある空気を送風して3‐4日成長させてからろ過した培養上澄みを用いることで始めた。
本発明のプロテアーゼ及びその変異体のプロテアーゼ活性を決定するために N-スクシニル-L-アラニルl-L-アラニル-L-プロリル-L-フェニル-p-ニトロアニリド (suc-AAPF-pNA)(N-succinyl-L-alanyl-L-alanyl-L-prolyl-L- phenyl-p-nitroanilide (suc-AAPF-pNA) )が測定された。使用された試薬溶液は:0.005% TWEEN(登録商標)-80 (Tris希釈緩衝液)を含む100 mM Tris/HCl, pH 8.6,; 10 mM CaCl2及び0.005% TWEEN(登録商標)-80 (Tris/Ca 緩衝液)を含む100 mM Tris緩衝液 pH 8.6,; 及びDMSO (suc-AAPF-pNA 原液)中の160 mM suc-AAPF-pNA (Sigma: S-7388)であった。 suc-AAPF-pNA 標準溶液を準備するために、 1 ml suc-AAPF- pNA 原液が100 ml Tris/Ca 緩衝液に加えられ、少なくとも10秒間良く混合された。アッセイは10 μlの希釈プロテアーゼ溶液を各ウエルに加え、その後すぐに190 μl 1 mg/ml suc-AAPF-pNA標準溶液加えることにより行われた。溶液は5秒の間混合され、動的モード(5分間に20回測定)での吸光度の変化がMTP リーダー、25°Cにおいて410 nmで測定された。プロテアーゼ活性はAU (活性= ΔOD min-1 ml-1)で表わされた。
各LAS及びLAS/EDTAの存在下に試験用プロテアーゼの培養後にLAS及びLAS/EDTA安定性が、AAPFアッセイを用いて決定された残留活性の関数として測定された。
試薬
ドデシルベンゼンスルホン酸塩、ナトリウム塩(=LAS): Sigma D-2525
TWEEN(登録商標)-80: Sigma P-8074
TRIS緩衝液(遊離酸): (Sigma T-1378); 6.35 gが約 960 ml の水で溶解された; 4N HCl によってpHが8.2に調整された。TRISの最終濃度は 52.5 mM.である。
TRIS緩衝液- 100 mM / pH 8.6 (100mM Tris/0.005% Tween(登録商標)-80)
TRIS-Ca 緩衝液, pH 8.6 (100mM Tris/lOmM CaC12/0.005% Tween(登録商標)-80)
使用機器:
平底MTPs (Costar No. 9017)
Biomek FX
ASYS Multipipettor
Spectramax MTP Reader
iEMS 培養器/シェーカー
Innova 4330培養器/シェーカー
Biohit マルチチャネル ピペット
BMG Thermostarシェーカー
0.063% LAS溶液がpH 8.2 の52.5 mM Tris 緩衝液で作られた。suc-AAPF-pNA希釈標準溶液が1 mlの100 mg/ml suc-AAPF-pNA原液(DMSO中)をpH 8.6の100 ml (100 mM) TRIS緩衝液に加えて作られた。上澄みを希釈するために平底プレートが希釈緩衝液で満たされ、一定量の上澄みが加えられ良く混合された。希釈率は成長用プレートのプロテアーゼコントロールの濃度に依った(AAPF活性)。望ましい濃度は80 ppmであった。10μlの希釈された上澄みが190 μl 0.063% LAS緩衝液/ウエルに加えられた。MTPがテープで覆われ数秒振動させ、そして培養器 (Innova 4230)に入れ45℃で60分間200 rpmで攪拌した。各ウエルの10 μl 混合物を190μl suc-AAPF-pNA標準溶液を含む新しいMTP に移して10分間培養後当初の活性(t=10分)を決定した。これらの溶液は良く混合され、そしてMTP Reader (25℃で5分間で20回測定)で測定された。
残留活性(%)=[t-60 の値]*100 / [t-10の値]
LAS/EDTA安定化法
代表的な陰イオン界面活性剤(LAS=線状アルキルベンゼンスルホン酸塩、ドデシルベンゼンスルホン酸ナトリウムーDOBS)及びジナトリウムEDTAの存在でのプロテアーゼ変異体の安定性が、規定の条件で培養後測定され、残留活性が、AAPFアッセイを用いて決定された。使用された試薬はドデシルベンゼンスルホン酸ナトリウム塩(DOBS, Sigma No. D- 2525), TWEEN(登録商標)―80 (Sigma No. P-8074)、ジナトリウムEDTA (Siegfried Handel No. 164599-02), HEPES (Sigma No. H-7523)、無ストレス緩衝液50 mM HEPES (11.9 g/1),
0.005% Tween(登録商標)-80), pH8.0, ストレス緩衝液:50mM HEPES(11.9g/l),
0.1% (重量/体積) DOBS (1 g/1), 10 mM EDTA (3.36 g/1), pH 8.0、参考プロテアーゼ及びプロテアーゼ変異体培養上澄みであって、200 - 400 μg/mlのタンパク質を含む。使用機器は希釈プレートとしてV- 又はU-底MTP (各々Greiner 651101及び650161)を使用し、無ストレス及びLAS/EPTA緩衝液のためにF-底MTP(Corning 9017)を使用、及びsuc- AAPF-pNA プレート, Biomek FX (Beckman Coulter)、Spectramax Plus 384 MTP Reader (分子機器)、Thermo Electron Corporation のiEMS培養器/シェーカー (1 mm振幅), シールテープ: Nunc (236366)であった。
D. 洗浄効果アッセイ
プロテアーゼ変異体の汚れ除去効果は市販の洗剤で決定された。市販の洗剤調合の熱不活性化はいずれのタンパク質成分の酵素活性を破壊するが、非酵素成分の特性は保持する。
このように、この方法は、市販洗剤を本発明の酵素変異体の試験で使用するのに適している。
円の直径が1/4インチのミクロスウォッチをCFTから得た。単一のミクロスウォッチ又は2つのミクロスウォッチが96ウエルMTPの各ウエルに垂直に置かれ、全表面領域が露出された(すなわち、ウエルの底に平らにではなく)。
このタイプのミクロスウォッチは非イオン水で室温において20分事前洗剤した。事前洗浄ステップの後に、スウォッチは紙タオルの上に置いて乾燥させた。空気乾燥されたスウォッチはエキスパルション プレス(expulsion press)上で1/4インチ円形金型を用いて打ち抜かれた。最終的に2つのミクロスウォッチが96ウエルMTPの各ウエルに垂直に置かれ全表面領域が露出された(すなわち、ウエルの底に平らにではなく)。
北米(NA)及び西ヨーロッパ(WE)の重質液体洗濯洗剤(HDL)について、事前に重量が計測された液体洗剤(ガラス瓶入り)を95℃の温浴水に2時間置いて熱不活性化が実施された。洗剤はその土地のスーパーマーケットで購入した。加熱しない及び加熱した洗剤の両方について洗剤を溶解後5分以内にアッセイし正確に非活性化されたパーセントを決定した。酵素活性はAAPFアッセイにより試験した。
参考セリンプロテアーゼ変異体のサンプルをMTPプレートで成長させた培養物のろ過培養ブロスから得た。使用機器はBiomek FX Robot (Beckman Coulter), SpectraMAX MTP リーダー (タイプ 340; Molecular Devices), iEMS 培養器 /シェーカー(Thermo/Labsystems); 平底MTPs (培養後に反応プレートを測定するために使用されるCostarタイプ9017)及びV-底MTP (上澄みの事前希釈のために使用したGreiner 651101)であった。このアッセイでは、プロテアーゼが基質を加水分解し基質から色素及び不溶粒子を遊離させる。このように濁りの割合は酵素活性の測定基準となる。
得られた吸光度がブランク値(酵素を持たない基質)に照らし訂正され、加水分解活性の測定基準を提供した。各サンプル(変異体)について、効果指数が計算された。効果指数は変異体の効果(実際の値)及び標準酵素(理論値)を同じタンパク質濃度で比較する。
さらに、理論値は標準酵素のラングミュア式のパラメータを用いて計算することができる。
プロテアーゼ変異体を区別するために、相対的特異活性がsuc-AAPF-pNA を基質として用いて計算された。これは変異体と野生型又は標準プロテアーゼの比較及びランク付けを可能にした。suc-AAPF-pNA 基質上の特異活性が、上に述べたアッセイを用いて、タンパク質分解活性を各サンプルの測定されたTCA値により除して決定された。これらの値を用いて相対的特異活性が計算された(変異体の特異活性/参考プロテアーゼの特異活性)。
効果指数は変異体の効果(実際値)と参考プロテアーゼの標準(理論値)を同じ濃度で比較するものである。さらに、理論値は標準プロテアーゼのラングミュアの式のパラメータを用いて計算することができる。1より大きい効果指数(PI)(PI>1)は標準と比べ(例えば、野生型)、より良い変異体を同定し、1のPI(PI=1)は標準と同じ効果の変異体を同定し、1より小さいPI(PI<1)は標準より悪い効果を奏する変異体を同定する。このように、PIは勝者を識別し、またある環境では使用するのに、より望ましくない変異体を識別する。
標的ISD(挿入置換欠失)ライブラリー構築
PCRベースによる方法が、図1に示すBPN'-Y217L (成熟領域の63-77及び92-132の位置)の2つの領域の、イン‐フレーム挿入、欠失及び/又は置換を含む修飾されたバチルス アミロリクエファシエンス スブチリシンBPN'-Y217L (PURAFECT(登録商標)PRIME スブチリシンとして市販されている)ポリヌクレオチドのライブラリーを作るために用いられた(Pisarchik 他、Prot. Eng. Des. Select., 20:257-265 [2007])を参照)。392アミノ酸の完全長タンパク質のBPN'-Y217L遺伝子の標的領域を均等にカバーする2組のオリゴヌクレオチドが正方向及び逆方向の両方に向けて、BPN'-Y217L遺伝子の部分の5'及び3'セグメントを増幅するのに使用された。BPN'-Y217L 成熟プロテアーゼのコード領域はクローンの目的のためにKpnl及びXhol制限部位を含む。
BPN'-Y217LプロテアーゼをコードするDNA配列を含む45ライブラリーにより形質転換された。細胞は1 ml のLuria Broth (LB)中で37℃で1時間成長させ、その後1.6% スキムミルク及び10 mg/1 ネオマイシンを含むLBプレートに平板培養のため移され、37℃で一夜培養された。各45ライブラリーから少なくとも2000クローンがハロー生成のためにスクリーニングされた。スキムミルク プレートに塗布されると0.05%から20%のクローンがハローを生成した。ハローを生成する限られた数のクローンが配列決定された。これらのクローンは挿入及び欠失の比率は種々であったが、フレームシフト変異を持つものはなかった。ハローを生成するクローンを実施例1に記載の、96ウエルプレートアッセイを用いてさらにAAPF活性のためにスクリーニングした。BPN'-Y217L 変異体タンパク質が上で述べた様に、96マイクロタイターウエルプレート中でバチルス スブチリス形質転換体を成長させて生成された。培養上澄みのタンパク質濃度が実施例1に記載のTCA 沈殿法により決定された。
標的化ISDにより生成されたBPN'-Y217L変異体の汚れ除去効果
汚れ除去活性(EMPA 116スウォッチ (BMI 汚れ, CFT)を用いて、pH 8、温度16°Cで洗濯での汚れ除去効果を決定するミクロスウォッチアッセイ及び上に述べた様に生成されたBPN'-Y217L変異体をTCA 沈殿法(関心の対象の特性についての試験)によりタンパク質が決定された)を決定するための実験の結果を表3−1に示す。結果は、実施例1に示す方法に、汚れ除去効果アッセイについて以下の変更を加えて実施して得た。
BPN' 3の組み合わせ変異体の構築
BPN'G97A-G128A-Y217Q (「BPN'3」)を親として用いて、組み合わせ変異体が、4つの遺伝子断片を用いて伸長(融合)PCRを使用するストラテジーにより作られた。断片1がP4974及びP4977 (表4-1を参照)プライマーにより増幅された。断片4がP4978及びP4976により増幅された(表4-1を参照)。断片1及び4の両方とも突然変異は含んでいなかった。各断片2及び3は、BPN'3をテンプレートとして、又はテンプレートが存在しない場合は一部重複する正方向及び逆方向プライマーを用いて、所望の突然変異を含む12及び16配列のセットとしてそれぞれ作られた。
02: P4981 及び P4982 (テンプレートなし)
03: P4983 及び P4984 (テンプレートなし)
04: P4985 及び P4986 (テンプレートなし)
05: P4987 及び P4988 (テンプレートなし)
06: P4989 及び P4990 (テンプレートなし)
07: P4991 及び P4992 (テンプレートなし)
08: P4993 及び P4994 (テンプレートなし)
09: P4995 及び P4996 (テンプレートなし)
10: P4997 及び P4998 (テンプレートなし)
11 : P4999 及び P5000 (テンプレートなし)
12: P5001 及び P5002 (テンプレートなし)
断片3の」配列のセットは以下のプライマーにより増幅された。
02: P5005 及び P5006 (テンプレートなし)
03: P5007 及び P5008 (テンプレートなし)
04: P5009 及び P5010 (テンプレートなし)
05: P5011 及び P5012 (テンプレートなし)
06: P5013 及び P5014 (テンプレートなし)
07: P5015 及び P5016 (テンプレートなし)
08: P5017 及び P5018 (テンプレートなし)
09: P5019 及び P5020 (テンプレートなし)
10: P5021 及び P5022 (テンプレートなし)
11:P5023 及び P5024 (テンプレートなし)
12: P5025 及び P5026 (テンプレートなし)
13: P5027 及び P5028 (テンプレートなし)
14: P5029 及び P5030 (テンプレートなし)
15: P5031 及び P5032 (テンプレートなし)
16: P5033 及び P5034 (テンプレートなし)
組み合わせ変異体の構築に使用されたプライマーを表4−1に示す。
融合PCR反応で生成された、完全長の断片がGel Band Purificationキット(Qiagen)により精製され、BamHI及びHindIII制限酵素によって消化され、バチルス発現プラスミドpHPLT-BPN partial opt (図2C)中で連結され、そして同じ制限酵素により切断された。
BPN' 3の組み合わせ変異体の汚れ除去効果
実施例4に記載の様に生成されたBPN' 3の組み合わせ変異体の汚れ除去活性が実施例3に記載の方法で試験された。
位置128,129及び130のBPN' 3の組み合わせ変異体の構築
BPN'G97A-G128A-Y217Q (BPN' 3)の位置128/129/130の回りに焦点をあてた組み合わせライブラリーが部位飽和特異突然変異及び/又は部位特異挿入及び/又は欠失を用いて作られた。生成された変異体は置換、挿入及び欠失変異を含んでいる。ライブラリーLl, L2 及びL3はWT位置S130S 及びL4 はS130Aに基づくものである。位置128/129及び130の回りに焦点を当てた4つの飽和ライブラリーが表6−1に示す様に構築された。
位置128, 129, 及び 130のBPN' 3組み合わせ変異体の汚れ除去効果
実施例6に記載の様に、位置128, 129, 及び 130のBPN' 3組み合わせ変異体の汚れ除去活性が実施例3に記載の様に試験された。各所で説明したように、BPN'変異体の機能が、変異体の、親タンパク質BPN'3に対する効果比率である、効果指数(「Pi」)として数値化された。BMI汚れ除去効果及び/又はTCA沈殿が0.5以上のPi値を示すBPN'3変異体は改善された洗剤効果及び/又は発現を示した。結果は表7−1に示す。
BPN'の組み合わせ変異体の構築
BPN'の組み合わせ変異体は、次のBPN'突然変異体--BPN' G97A/G128A/Y217Q, BPN' M124V/L126A/Y217Q, BPN' G128A/Y217Q及びBPN' N123G/Y217Qを親分子として使用する伸長(融合)PCRにより、位置96, 98, 129及び/又は222に挿入又は置換を作ることで作られた。各作られた組み合わせ突然変異体について、用いられた親DNA分子、導入された突然変異及びPCRプライマーは表8−1に示す。
BPN'3組み合わせ変異体の汚れ除去効果
実施例8に記載の様に生成されたBPN'3組み合わせ変異体の汚れ除去活性が実施例3に記載の様に試験された。各所で説明したように、BPN'変異体の機能が、変異体の、タンパク質BPN'3(BPN' G97A-G128A-Y217Q)に対する比率である効果指数(「Pi」)として数値化された。BMI汚れ除去効果及び/又はTCA沈殿が0.5以上のPi値を示す変異体は改善された洗剤効果及び/又は発現を示した。表では0.05以下の効果指数は0.05に固定され、太字斜字体で示す。
Claims (16)
- 置換Y217L及びさらに請求項1乃至4の何れか1項に記載の少なくとも1つの修飾セットを含むスブチリシン変異体であって、前記位置が配列番号2のBPN’スブチリシンの位置に対応する、前記スブチリシン変異体。
- 置換G97A/G128A/Y217Q及びさらに請求項1乃至4の何れか1項に記載の少なくとも一つの修飾セットを含むスブチリシン変異体であって、前記位置が配列番号2のBPN’スブチリシンの位置に対応する、前記スブチリシン変異体。
- 請求項1乃至6の何れか1項に記載の少なくとも一つのスブチリシン変異体を含む洗浄組成物。
- 前記洗浄組成物が洗濯用洗剤である、請求項7の洗浄組成物。
- 前記洗濯用洗剤が重質液体洗濯洗剤である、請求項8の洗濯用洗剤。
- 前記洗浄組成物が食器洗い用洗剤である、請求項7の洗浄組成物。
- 洗浄組成物が以下の群、すなわち、
ヘミセルラーゼ、ペルオキシダーゼ、プロテアーゼ、セルラーゼ、キシラナーゼ、リパーゼ、フォスフォリパーゼ、エステラーゼ、クチナーゼ、ペクチナーゼ、ケラチナーゼ、レダクターゼ、オキシダーゼ、フェノール オキシダーゼ、リポオキシダーゼ、リグリナーゼ、プルラナーゼ、タンナーゼ、ペントサナーゼ、マラナーゼ、β‐グルカナーゼ、アラビノシダーゼ、ヒアルニダーゼ、コンドロイチナーゼ、ラッカーゼ、及びアミラーゼ又はこれらの混合物から選択される一以上の追加の酵素又は酵素誘導物をさらに含む、請求項7乃至10の何れか1項に記載の洗浄組成物。 - さらに少なくとも一つの安定化剤を含む、請求項7乃至11の何れか1項に記載の洗浄組成物。
- 請求項1乃至7の何れか1項に記載の少なくとも一つのスブチリシン変異体を少なくとも0.0001重量%で含み、及び任意選択的に少なくとも一つの好適な付加成分を含む、前記洗浄組成物。
- 洗浄方法であって、前記方法は
a) 物の表面及び/又は織物を含む商品を請求項7乃至13の何れか1項に記載の洗浄組成物に接触させる工程、及び
b)任意選択的に前記表面及び商品を洗浄し及び/又はすすぐ工程、
を含む、洗浄方法。 - 請求項1乃至7の何れか1項に記載の少なくとも一つのスブチリシン変異体を含む、動物用飼料。
- 請求項1乃至7の何れか1項に記載の少なくとも一つのスブチリシン変異体を含む食品加工用組成物。
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