JP5893584B2 - 1以上の組み合わせ変異を含むプロテアーゼ - Google Patents
1以上の組み合わせ変異を含むプロテアーゼ Download PDFInfo
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Description
101Q-G118Q-T213Q-L217Eから選択された置換の組み合わせを含み、その位置は、配列番号1として記載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’のアミノ酸配列との対応によって番号を付けられる。ある実施態様では、この洗浄組成物は洗剤である。ある実施態様では、この洗剤は食器洗剤である。他の実施態様では、この洗剤はランドリー洗剤、たとえば重質の液状または乾燥ランドリー洗剤である。あるいは他の実施態様では、該洗浄組成物は少なくとも1の安定剤をさらに含む。
8R-K213R, S24E-A45E-S101E-N109E-N118E-K213R, S24E-A45E-S101E-N109E-N118E-K213E, S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217QおよびS24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Eから選択された置換の組み合わせを含み、その位置は配列番号1のBPN’ スブチリシンの位置に対応する。ある実施態様では、この洗浄組成物は洗剤である。他の実施態様では、この洗剤は食器洗剤である。さらに他の実施態様では、この洗剤はランドリー洗剤、たとえば重質の液状または乾燥ランドリー洗剤である。あるいは他の実施態様では、該洗浄組成物は少なくとも1の安定剤をさらに含む。
101Q-G118Q-T213Q-L217Eから選択された置換の組み合わせを含み、その位置は、配列番号1として記載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’のアミノ酸配列との対応によって番号を付けられ、かつ該スブチリシン変異体は1以上の追加の酵素または酵素誘導体をさらに含む。この追加の酵素または酵素誘導体は、ヘミセルラーゼ、ペルオキシダーゼ、プロテアーゼ、金属プロテアーゼ、セルラーゼ、キシラナーゼ、リパーゼ、ホスホリパーゼ、エステラーゼ、ペルヒドロラーゼ、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、リポキシゲナーゼ、リグニナーゼ、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、ヒアルロニダーゼ、コンドロイチナーゼ、ラッカーゼおよびアミラーゼ、またはこれらの混合物から選択される。ある実施態様では、この洗浄組成物は洗剤である。ある実施態様では、この洗剤は食器洗剤である。他の実施態様では、この洗剤はランドリー洗剤、たとえば重質の液状または乾燥ランドリー洗剤である。あるいは他の実施態様では、該洗浄組成物は少なくとも1の安定剤をさらに含む。
S24E-A45E-S101E-N109E-N118E-K213R, S24E-A45E-S101E-N109E-N118E-K213E, S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217QおよびS24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Eから選択された置換の組み合わせを含み、その位置は配列番号1のBPN’スブチリシンの位置に対応し、かつ該スブチリシン変異体は1以上の追加の酵素または酵素誘導体をさらに含む。この追加の酵素または酵素誘導体は、ヘミセルラーゼ、ペルオキシダーゼ、プロテアーゼ、金属プロテアーゼ、セルラーゼ、キシラナーゼ、リパーゼ、ホスホリパーゼ、エステラーゼ、ペルヒドロラーゼ、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、リポキシゲナーゼ、リグニナーゼ、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、ヒアルロニダーゼ、コンドロイチナーゼ、ラッカーゼおよびアミラーゼ、またはこれらの混合物から選択される。ある実施態様では、この洗浄組成物は洗剤である。他の実施態様では、この洗剤は食器洗剤である。さらに他の実施態様では、この洗剤はランドリー洗剤、たとえば重質の液状または乾燥ランドリー洗剤である。あるいは他の実施態様では、該洗浄組成物は少なくとも1の安定剤をさらに含む。
217QおよびS24Q-R45Q-S101Q-G118Q-T213Q-L217Eから選択された置換の組み合わせを含み、その位置は、配列番号1として記載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’のアミノ酸配列との対応によって番号を付けられる。
N109E-N118R-K213R, S24E-A45E-S101E-N109E-N118E-K213R, S24E-A45E-S101E-N109E-N118E-K213E, S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217QおよびS24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Eから選択された置換の組み合わせを含み、その位置は配列番号1のBPN’スブチリシンの位置に対応する。
定義
I.プローブタンパク質のアッセイ
II.物理的特性最適値の決定
III.物理的特性最適値を有する変異体タンパク質の準備
セリンプロテアーゼ酵素の応用
洗浄組成物
テアーゼが国際公開第95/23221号、国際公開第92/21760号、米国特許出願公開第2008/0090747号、および米国特許第5,801,039号、5,340,735号、5,500,3645号、5,855,625号、米国再発行特許第34,606号、米国特許第5,955,340号、5,700,676号、6,312,936号および6,482,628号ならびに各種の他の特許に記載されている。ある実施態様では、金属プロテアーゼ、たとえば国際公開第07/044993号に記載された中性金属プロテアーゼがこれに限定されることなく本発明に用いられる。
実験
以下の実施例では、読むのが楽なように以下に記載された様々なアッセイが用いられた。以下に提示される手順から外れる個所はいずれも明示される。
A.96ウェルマイクロタイタープレートにおけるタンパク質含量測定のためのTCAアッセイ
B.洗浄性能アッセイ
染み除去活性の計算
性能指数
この実施例では、GG36(本明細書ではバチルスレンタススブチリシンとも呼ばれる。)を産生するために実施された実験が記載される。形質転換は従来技術で知られているように実施された(たとえば、国際公開第02/14490号参照)。
バチルススブチリス中でのGG36プロテアーゼの産生
バチルスレンタススブチリシン(=GG36)の組み換え電荷/疎水性ライブラリー(CCHL)の設計および生成
プロテアーゼ変異体の発現
バチルススブチリス中でのFNAプロテアーゼの産生
この実施例では、バチルススブチリス中でFNA(本明細書ではバチルススブチリススブチリシンBPN'-Y217L (=FNA)とも呼ばれる。)を産生するために実施された実験が記載される。形質転換は従来技術で知られているように実施された(たとえば、国際公開第02/14490号参照)。
プロテアーゼ変異体の発現
この実施例は、BMIマイクロスウォッチアッセイにおけるGG36およびFNA組み換え疎水性ライブラリー変異体の試験を記載する。実施例1に提示された方法が用いられた。表4−1(GG36)および表4−2(FNA)に示された結果は、相対タンパク質発現(TCA PI)および染み除去活性(BMI PI)について、これらの変異体の性能がそれぞれの親と比較される性能指数を表す。0.05以下の性能指数は0.05とされ、太字イタリック体で示された。NDは検出されなかったことを示す。
Claims (8)
- バチルススブチリシン(Bacillus subtilisin)のスブチリシン変異体を含んでいる洗剤組成物であって、当該スブチリシン変異体が、タンパク質分解活性を有し2以上の位置におけるQ置換を有する成熟型であり、当該バチルススブチリシンがFNAであり、前記変異体が、S24Q-A45Q-S101Q-N109Q-N118Q-K213Q, A45Q-S101Q-N109Q-N118Q-K213Q, S101Q-N109Q-N118Q-K213Q, N109Q-N118Q-K213Q, N118Q-K213Q, S24E-A45Q-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101Q-N109Q-N118Q-K213Q, S24E-A45E-S101E-N109Q-N118Q-K213Q, S24E-A45E-S101E-N109E-N118Q-K213Q, S24L-A45Q-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101Q-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109Q-N118Q-K213Q, S24L-A45L-S101L-N109L-N118Q-K213Q, S24R-A45Q-S101Q-N109Q-N118Q-K213Q, S24R-A45R-S101Q-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109Q-N118Q-K213Q, S24R-A45R-S101R-N109R-N118Q-K213Q, S24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217QおよびS24Q-A45Q-S101Q-N109Q-N118Q-K213Q-L217Eから選択される2以上の位置における置換を有する変異体であり、当該位置が配列番号1のBPN’スブチリシンの位置に対応している、洗剤組成物。
- 当該スブチリシン変異体が、その親酵素と比較して、0.5以上である相対タンパク質発現レベル性能指数(TCA PI)および/または染み除去活性性能指数(BMI PI)を有する、請求項1に記載の洗剤組成物。
- 当該洗剤が重質の液状または乾燥ランドリー洗剤である、請求項1に記載の洗剤組成物。
- 当該洗剤が食器洗剤である、請求項1に記載の洗剤組成物。
- ヘミセルラーゼ、セルラーゼ、ペルオキシダーゼ、プロテアーゼ、金属プロテアーゼ、キシラナーゼ、リパーゼ、ホスホリパーゼ、エステラーゼ、ペルヒドロラーゼ、クチナーゼ、ペクチナーゼ、ペクチン酸リアーゼ、マンナナーゼ、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、リポキシゲナーゼ、リグニナーゼ、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、ヒアルロニダーゼ、コンドロイチナーゼ、ラッカーゼおよびアミラーゼもしくはこれらの混合物から選択された1以上の追加の酵素または酵素誘導体をさらに含む、請求項1〜4のいずれか1項に記載の洗剤組成物。
- 少なくとも1の安定剤をさらに含む、請求項1〜5のいずれか1項に記載の洗剤組成物。
- 少なくとも1の当該スブチリシン変異体を少なくとも0.0001重量パーセント、および任意的な少なくとも1の適当な補助成分を含んでいる、請求項1〜6のいずれか1項に記載の洗剤組成物。
- 洗浄する方法であって、当該方法が
a)表面および/または布地を含む物品を請求項1〜7のいずれか1項に記載の洗剤組成物と接触させる工程、および
b)任意的に、当該表面もしくは物品を洗いおよび/またはすすぐ工程
を含む、洗浄する方法。
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US61/218,802 | 2009-06-19 |
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JP2013120314A Expired - Fee Related JP5893584B2 (ja) | 2008-11-11 | 2013-06-07 | 1以上の組み合わせ変異を含むプロテアーゼ |
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CA (1) | CA2743302A1 (ja) |
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Cited By (1)
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JP2015015952A (ja) * | 2008-11-11 | 2015-01-29 | ダニスコ・ユーエス・インク | 1以上の組み合わせ変異を含むプロテアーゼ |
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US20150065412A1 (en) | 2015-03-05 |
JP2013236632A (ja) | 2013-11-28 |
AR074311A1 (es) | 2011-01-05 |
US9096838B2 (en) | 2015-08-04 |
CN103361196B (zh) | 2016-01-27 |
CN106399280B (zh) | 2020-12-22 |
US20160010072A1 (en) | 2016-01-14 |
JP2015015952A (ja) | 2015-01-29 |
RU2560978C2 (ru) | 2015-08-20 |
WO2010056653A3 (en) | 2010-09-16 |
JP6120815B2 (ja) | 2017-04-26 |
EP2650354A2 (en) | 2013-10-16 |
MX2011004802A (es) | 2011-06-16 |
CN106399280A (zh) | 2017-02-15 |
BR122013014157A2 (pt) | 2018-11-13 |
US20140308736A1 (en) | 2014-10-16 |
WO2010056653A2 (en) | 2010-05-20 |
BRPI0922083A2 (pt) | 2017-05-30 |
CN103361196A (zh) | 2013-10-23 |
EP2650354A3 (en) | 2014-02-12 |
CN102209775A (zh) | 2011-10-05 |
CN103122345A (zh) | 2013-05-29 |
KR20110095260A (ko) | 2011-08-24 |
EP2362896A2 (en) | 2011-09-07 |
RU2011123739A (ru) | 2012-12-20 |
EP3031894A1 (en) | 2016-06-15 |
US20120003718A1 (en) | 2012-01-05 |
JP2012508031A (ja) | 2012-04-05 |
CA2743302A1 (en) | 2010-05-20 |
US8753861B2 (en) | 2014-06-17 |
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