JP6522570B2 - 生物機能材料 - Google Patents
生物機能材料 Download PDFInfo
- Publication number
- JP6522570B2 JP6522570B2 JP2016207807A JP2016207807A JP6522570B2 JP 6522570 B2 JP6522570 B2 JP 6522570B2 JP 2016207807 A JP2016207807 A JP 2016207807A JP 2016207807 A JP2016207807 A JP 2016207807A JP 6522570 B2 JP6522570 B2 JP 6522570B2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
- C09D5/14—Paints containing biocides, e.g. fungicides, insecticides or pesticides
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
- C09D5/16—Antifouling paints; Underwater paints
- C09D5/1606—Antifouling paints; Underwater paints characterised by the anti-fouling agent
- C09D5/1637—Macromolecular compounds
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
- C09D5/16—Antifouling paints; Underwater paints
- C09D5/1687—Use of special additives
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Description
本発明は、自浄性組成物、ならびに鳥の糞、虫の死骸、食べかす及びほかの染みの原因となる物質による表面のステイン沈着を防止及び減少させるための方法に関する。
自動車の内部及び外部表面、例えばコーティング、ペイント、及びシート布はともに、鳥の糞、昆虫の死骸、針葉樹の樹脂、微生物、ガム等に長期にわたり暴露されると汚染及び腐食を受ける。一定のステイン(stain)、例えば昆虫由来のステインは、通常の自動ブラシを使用しない洗浄で除去することは困難である。内部表面及びコーティングもまた、食品及び飲料の油、タンパク質、糖類、及びほかの成分によって容易に染色される恐れがあり、このようなステインの適時な除去には一定の努力が必要である。
コーティング又は表面を抗菌剤、抗真菌剤もしくは防汚剤を伴う表面を供するための基質中に酵素を混入することが知られている。しかしながら、出願人が知る限り、表面に接触するステイン分子を酵素的に分解する目的で表面に消化タンパク質を付着することは新規である。
第1の観点において、本発明は、基質、ステイン分子を分解することが可能な消化タンパク質、及びリンカー分子を含んでなる組成物を供する。
第1の観点において、本発明は、基質、ステイン分子を分解することが可能な消化タンパク質、及びリンカー分子を含んでなる組成物を供する。本発明は、具体的には、表面、例えばペイント及びコーティング上における、リゾチーム、プロテアーゼ、リパーゼ、セルラーゼ等を含む1又は複数の消化酵素の混入に関する。消化タンパク質の触媒活性は、ステイン汚染を減少及び除去するための自己浄化(self-cleaning)を可能にする。多様なステインは、虫の破損した死骸、動物(鳥等)の排泄物、食品及びほかの飲料、ならびに化粧品及びパーソナルケア製品から形成されるものを含む。詳細な成分はステイン源によって変化するが、表面に付着するステインの主要な成分は、タンパク質、多糖、脂質又は油である。
酵素はプラスチックの表面に付着させることができる。〜100%の表面被覆率に相当する酵素付着は、100〜1000nmの範囲の直径を有するポリスチレン粒子により達成することができる。消化タンパク質でコーティングすることによりこれらの粒子を、物質の表面を機能的にするためにペイント又はコーティングと一緒に使用してもよい。同様の化学的な結合アプローチは、予め形成されたプラスチック部品上に酵素をコーティングするために適用することができ、これにより部品の表面上にタンパク質コーティングが形成される。図1に示されるとおり、100nm〜1000nmの直径を有する粒子は、エマルション重合により合成することができる。一般的には、エマルション重合は、水、モノマー、及び界面活性剤を含むエマルション中で行われる重合の一種である。エマルション重合の最も一般的なものは、モノマー(油)の液滴が水の連続相中で(界面活性剤により)乳化される水中油エマルションである。
ステインは異なる接触源から生じうる。虫の死骸、動物の排泄物、食品、ミルク及びほかの飲料、ならびに化粧品及びパーソナルケア製品は全てステインの原因となる。詳細な成分はステイン源によって変化するが、表面に付着するステインの主要な成分は、タンパク質、単糖、多糖、脂質又は油である。消化タンパク質、例えばリパーゼ、プロテアーゼ、アミラーゼ及びセルラーゼ(これらはそれぞれ異なる成分を攻撃する)は、このようなステインに対抗するために、極めて有効、安全かつ経済的な薬剤である。表1下に示されるとおり、これらのタンパク質を、出願人のスクリーニングテストにおいて調査し試験したところ、出願人は、活性測定における容易さにより、その後の実験の大半を追行するためにプロテアーゼを最終的に選択した。
N−アクリルオキシスクシンイミド(392mg)、1.2mlのスチレン及び29.2mgの4,4’−アゾビス−(シアノバレリアン酸)を16mlのクロロホルムとともに20mlのガラス反応バイアル中で混合した。バイアルを窒素でパージし、密封し、そして攪拌しながら70℃で12時間インキュベートし、続いてパージしている窒素により溶媒を除去した。ポリマー生成物を50mg/mlの濃度においてクロロホルム中に再度溶解させた。1mlの生じた溶液を6000rpmにおいてポリスチレンプレート(11cm直径)上にスピンコーティングした。Subtilisin Carlsberg由来のプロテアーゼを10mg/mlの濃度において0.05Mリン酸緩衝液中に溶解させた。酵素を以下の3ステップ多層スピンコーティングを介して活性ポリマーでコーティングしたプレート上に適用した:1)1mlのプロテアーゼ溶液、2)0.5%(V/V)のグルタルアルデヒドを含有する1mlのプロテアーゼ溶液、3)1mlのプロテアーゼ溶液。スピンコーティングしたプレートを4℃で12時間保ち、その後0.05Mのトリス緩衝液(pH8)、2M NaCl溶液及びDI水で全面的に洗浄した。最後に、該プレートを空気乾燥させ、そして小片(1×2cm)にカットした。この方法は共有結合架橋と称した。比較として、同様の手順を活性ポリマーコーティングを伴わないポリスチレンプレート上に適用し、これを物理的吸着と称した。
酵素コーティングの視覚化
まず、蛍光染料(オレゴングリーン, Invitrogen Corp.)を2mg/mlの濃度においてジメチルスルホキシドに溶解させた。物理的に吸着した酵素及び共有結合的に固定化した酵素を伴う試料プレートを、染料溶液中で静かに振とうさせながら室温で2時間インキュベートし、続いてDI水でリンスした。その後該プレート窒素中で乾燥させ、蛍光顕微鏡で観察した。そのイメージを図2に示す。ここで緑色は酵素で覆われた領域を示す。物理的吸着と比較して、共有結合架橋法を使用した場合、極めて多量の酵素が表面上に固定化した。
酵素積載の測定
プラスチックプレートに付着した酵素の量を、改良したブラッドフォード法により測定した。典型的には、まず、ブラッドフォード試薬をDI水で希釈(1:5、体積)することにより希釈標準溶液を調製した。標準として遊離プロテアーゼを使用して較正曲線が得られた。1mlのキュベット中、0.5mlのプロテアーゼ溶液を0.5mlの希釈標準溶液と混合し、その後5分間反応させた。該溶液の吸光度を分光光度計において465nmで測定した。一連の異なるプロテアーゼ濃度を試験した後、図3に示される較正曲線が得られた。
酵素コーティングのタンパク質分解活性の検証
溶液中の酵素:基質として0.65%(w/v)カゼインを使用してプロテアーゼのタンパク質分解活性を測定した。プロテアーゼ溶液(0.1ml)を0.5mlのカゼイン溶液とともに37℃で10分間インキュベートした。0.5mlのトリクロロ酢酸(110mM)を添加することにより反応を停止させた。該混合物を遠心分離し、沈殿物を除去した。生じた上清(0.4ml)を1mlの炭酸ナトリウム(0.5M)及び0.2mlのDI水で希釈したフォリン・シオカルト(Folin and Ciocalteu)フェノール試薬(フォリン・シオカルトフェノール試薬をDI水で1:4に希釈)と混合し、続いて37℃で30分間インキュベートした。最後に、該混合物を再度遠心分離し、そして上清の吸光度を分光光度計において660nmで測定した。100μlの緩衝液を添加し、そして同様の試験を行うことにより酵素溶液を伴わないブランク実験を行った。ブランクの吸光度は試料(酵素溶液)から差し引いた。
酵素コーティングにおけるステイン分解
モデルステインとして卵白を使用して、酵素コーティング上におけるステイン分解を測定した。プロテアーゼコーティングを伴うプレート(11cmの直径)において、2mlの卵白溶液(DI水中10mg/ml)を2000rpmでスピンコーティングした。その後プレートを小片(1×2cm)にカットし、そして様々な時間において、室温(25℃)に保ち、卵白を分解させた。一定の時間後、1つの小片をDI水で注意深く洗浄し、そして洗浄溶液中の卵白を、ゲル透過クロマトグラフィー(GPC)を使用して分析し、分子量の変化を測定した。該GPCクロマトグラフ中には、典型的な2つのピークが見られた:1方は短い保持時間を有し、そして他方は長い保持時間を有し、これらはそれぞれ卵白と分解生成物に対応する。卵白ピークの面積に基づき、図6に示される卵白分解の経時変化を得た。また、プロテアーゼコーティングを伴わないプレートを用いて対照実験を行ったが、明確な生成物ピークは同定されなかった。
酵素コーティングの熱安定性
酵素コーティングの熱安定性を、空気加熱オーブン内にて80℃で実験した。一定の時間間隔で試料プレートをオーブンから取り出し、そして例2に記載される手順に従い活性を測定した。時間による活性の減少を図9に示す。共有結合架橋酵素は、物理的吸着酵素と比較して、熱不活性化に対して優れた安定性を示す。
Claims (2)
- 自浄性材料を形成するための方法であって、
チオール基、カルボキシル基、酸無水物基及びエステル基からなる群から選択される官能基を含む基質を表面に結合させる工程、ここで、前記官能基は、前記基質の外部表面に結合する;及び
前記基質上に消化タンパク質をスピンコーティングし、これにより消化タンパク質の活性基と前記官能基との間にリンカー部分を形成する工程、ここで、前記消化タンパク質は、リゾチーム、プロテアーゼ、リパーゼ、セルラーゼ、グリコシダーゼ、及びアミラーゼから成る群から選択される、
を含み、
ここで、前記タンパク質と前記基質との間の前記リンカー部分は、前記タンパク質を前記基質の外部表面に共有的に連結させ、そして、前記消化タンパク質は、前記基質の表面上で飽和し、ステインとの反応に曝されることを特徴とする、方法(ただし、前記自浄性材料を形成するための方法は、消化タンパク質が基質に組み込まれている自浄性材料を形成するための方法を除く)。 - 前記表面が、金属、ガラス、硬化ペイント、プラスチック、及び布から成る群から選択される、請求項1に記載の方法。
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US20180044658A1 (en) | 2018-02-15 |
EP2087165A4 (en) | 2009-12-02 |
US20190153423A1 (en) | 2019-05-23 |
US10781438B2 (en) | 2020-09-22 |
JP6845268B2 (ja) | 2021-03-17 |
EP2087165A2 (en) | 2009-08-12 |
US20200342098A1 (en) | 2020-10-29 |
US9828597B2 (en) | 2017-11-28 |
JP6096748B2 (ja) | 2017-03-15 |
US20190153422A1 (en) | 2019-05-23 |
JP2010510380A (ja) | 2010-04-02 |
US11236323B2 (en) | 2022-02-01 |
JP2021101015A (ja) | 2021-07-08 |
WO2008063902A2 (en) | 2008-05-29 |
US20080119381A1 (en) | 2008-05-22 |
JP2017071779A (ja) | 2017-04-13 |
EP2087165B1 (en) | 2015-09-02 |
JP2015096610A (ja) | 2015-05-21 |
WO2008063902A3 (en) | 2008-11-13 |
US11225654B2 (en) | 2022-01-18 |
JP2019108550A (ja) | 2019-07-04 |
US20190153424A1 (en) | 2019-05-23 |
CN101600835B (zh) | 2014-01-22 |
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