JP2022500067A - 生体ナノ触媒固定化用のプリント可能な磁性粉末及び3dプリント対象物 - Google Patents
生体ナノ触媒固定化用のプリント可能な磁性粉末及び3dプリント対象物 Download PDFInfo
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Abstract
Description
本出願は、本明細書において参照によりその全体が援用される、2018年9月27日に出願された米国仮特許出願第62/737,910号明細書に係る利益を主張する。
材料:ポリアミド12(ナイロン12、カタログ番号AdSint PA12)及びポリスチレン(カタログ番号Coathylene PS Sint)を、Advanc3D Materials GmbH(Hamburg,Germany)から購入した。ポリプロピレン(カタログ番号Polyaxis PD 2000)サンプルを、A.Schulman(Fairlawn,Ohio)から入手した。10μmよりも小さい粒子サイズを有する酸化鉄(カタログ番号QM−D50/4マグネタイト)を、Reade Advanced Materials(East Providence,RI)から、及び5μmよりも小さい粒子サイズの酸化鉄(II、III)をSigma Aldrich(カタログ番号310069−500G,St Louis,MO)から入手した。用いられる発泡剤は、重炭酸ナトリウム(Sigma Aldrich、カタログ番号SX0320−1)及びクエン酸(VWR,Radnor,Pennsylvania、カタログ番号BDH8003−500G)であった。Fisher Scientific製のジメチルスルホキシド(DMSO、カタログ番号D128−4、Hampton,NH)をさらに精製せずに用いた。
熱可塑性プラスチック−マグネタイト複合体の3Dプリンティングは、Sintratec(著作権)Kit(https://sintratec.com/,Switzerland)を用いて実施した。対象物を、Autodesk Inventor(https://www.autodesk.com/)によりコンピュータモデリングした。ポリアミド12(ナイロン12、AdSint PA12)を、Advanc3D Materials GmbHから購入した。ポリプロピレン(Polyaxis,Fairlawn,Ohio、カタログ番号PD2000)サンプルを、A.Schulmanから入手した。10μmよりも小さい粒子サイズを有する酸化鉄(カタログ番号QM−D50/4マグネタイト)をReade Advanced Materialsから及び5μmよりも小さい粒子サイズの酸化鉄(II、III)をSigma Aldrich(カタログ番号310069−500G)から入手した。用いられる発泡剤は、重炭酸ナトリウム(EMD Millipore、カタログ番号SX0320−1)であった。2つのタイプの低密度ヒュームドシリカを、Cabot(CAB−O−SIL TS−530、CAB−O−SIL TS−610)から入手した。追加のタイプのシリカを、Evonik(AEROSIL R812)から入手した。多数の特性、特に高いヤング率、解像度、空隙率、降伏強度、及び磁性モーメントを低い弾性域及び疎水性で最適化した。プリンティングパラメータ(粉末流動性、チャンバ温度、プリントベッド温度及びレーザ速度)を調査し、対象物強度を維持しながら最大表面積を確保した。
a.110℃のチャンバ温度、125℃の表面温度、650mm/sのレーザ速度におけるポリプロピレン(46%w/w)、マグネタイト(<10μm 46%w/w)、及び重炭酸ナトリウム(8%w/w)の押出複合体ブレンド
b.110℃のチャンバ温度、125℃の表面温度、350mm/sのレーザ速度におけるポリプロピレン(48%w/w)、マグネタイト(<10μm 48%w/w)、及び重炭酸ナトリウム(4%w/w)の押出複合体ブレンド
c.119℃のチャンバ温度、129℃の表面温度、350mm/sのレーザ速度におけるポリプロピレン(36%w/w)、マグネタイト(<10μm 56%w/w)、及び重炭酸ナトリウム(8%w/w)の押出複合体ブレンド
a.140℃のチャンバ温度、164℃の表面温度、300mm/sのレーザ速度におけるナイロン12(49.875%w/w)、49.875%の[2%のBC、98%のマグネタイト<10μm](w/w)、及びシリカ(TS−610 0.25%w/w)の物理ブレンド
b.140℃のチャンバ温度、160℃の表面温度、200mm/sのレーザ速度における99.75%の[ナイロン12(50%w/w)、50%の[BC(2%w/w)、マグネタイト(<10μm 98%ww)]の溶解コーティング]、シリカ(TS−610 0.25%w/w)の物理ブレンド
c.140℃のチャンバ温度、170℃の表面温度、300mm/sのレーザ速度における79.75%の[マグネタイト(<10μm 46%w/w)、ナイロン12(46%w/w)、及び重炭酸ナトリウム(8%w/w)の押出複合体ブレンド]、ナイロン12(20%w/w)、シリカ(TS−610 0.25%w/w)の物理ブレンド。
ρcomposite=mC/vC=(mP+mM)/(vP+vM)(式1)
を用いて計算した。
%多孔度=100×[1−(ρsm/ρcomposite)](式2)
焼結材料上のインベルターゼ固定化:パン酵母由来のインベルターゼを、Sigma Aldrich(St.Louis,MO,USA、カタログ番号I4504)から購入した。すべての水を、BarnStead Nanopure水精製装置(Thermo Scientific、18.2Mオーム−cm)から入手した。酸化鉄ナノ粒子(NP)を既述のとおり調製し(各々参照によりその全体が本明細書において援用される米国特許第9,597,672号明細書;国際公開第2018102319号パンフレット)、pH11において4℃でN2スパージ雰囲気下で保管した。
大腸菌(E.coli)中で発現させた組換えBM3シトクロムp450、組換えグルコース脱水素酵素(GDH)、ウシ血清アルブミン(BSA)、ウシ肝カタラーゼ(CAT)、ウシ赤血球細胞質スーパーオキシドジスムターゼ(SOD)、グルコース(ベータ−d−グルコース)、p−ニトロフェニルラウリン酸塩(p−NPL)、p−ニトロフェノール(p−NP)、ニコチンアミドアデニンジヌクレオチドリン酸(還元)四ナトリウム塩(NADPH)を、3Dプリント足場上で固定化する。すべてのストック溶液を、Barnstead(商標)Nanopure(商標)水精製装置(Thermo Scientific)により精製された18.2MΩ−cm水を用いて形成した。吸光度を、Costar(商標)3635 UV透過マイクロプレート中でGen5(商標)ソフトウェアで操作するBiotek Synergy4(商標)プレートリーダを用いて三重で計測する。Fisher Scientific(登録商標)(Waltham,MA)製の1/8インチプローブを有する超音波処理装置(FB−505)を、すべての超音波処理に用いる。酸化鉄ナノ粒子(NP)を既述のとおり調製し(各々参照によりその全体が本明細書において援用される米国特許第9,597,672号明細書;国際公開第2018102319号パンフレット)及び各々参照によりその全体が本明細書において援用されるUniversal enzyme immobilization within hierarchically−assembled magnetic scaffolds.Catalysis & Biocatalysis 2016;34)、pH11において4℃でN2スパージ雰囲気下で保管した。
フローセル用に設計された3Dプリント足場は、インベルターゼを固定化する。すべての水を、BarnStead Nanopure水精製装置(Thermo Scientific、18.2Mオーム−cm)から入手した。酸化鉄ナノ粒子(NP)を既述のとおり調製し(各々参照によりその全体が本明細書において援用される米国特許第9,597,672号明細書;国際公開第2018102319号パンフレット)、pH11において4℃でN2スパージ雰囲気下で保管した。
Vmax/F=[S]oX−Km*ln(1−X)
焼結材料上で固定化されたHRPは、その活性を保持する。ホースラディッシュペルオキシダーゼ(HRP)(カタログ77332)を、Sigma Aldrich(St.Louis,MO,USA)から購入した。すべての水を、BarnStead Nanopure水精製装置(Thermo Scientific、18.2 Mオーム−cm)から入手した。酸化鉄ナノ粒子(NP)を既述のとおり調製し(Corgie SC,Brooks RT,Chairil R,Chun MS,Xie B,Giannelis EP.Universal enzyme immobilisation within hierarchically−assembled magnetic scaffolds.Catalysis & Biocatalysis 2016;34)、pH11において4℃でN2スパージ雰囲気下で保管した。
固定化HRPは、比色アッセイにより判定されるとおり3Dプリントフローセル中でその活性を保持する。ホースラディッシュペルオキシダーゼ(HRP)(カタログ77332)を、Sigma Aldrich(St.Louis,MO,USA)から購入した。すべての水を、BarnStead Nanopure水精製装置(Thermo Scientific、18.2Mオーム−cm)から入手した。酸化鉄ナノ粒子(NP)を既述のとおり調製し(Corgie SC,Brooks RT,Chairil R,Chun MS,Xie B,Giannelis EP.Universal enzyme immobilisation within hierarchically−assembled magnetic scaffolds.Catalysis & Biocatalysis 2016;34)、pH11において4℃でN2スパージ雰囲気下で保管した。
エピメラーゼを、フローセルとして設計された3Dプリント足場上に固定化した。すべての水を、BarnStead Nanopure水精製装置(Thermo Scientific、18.2Mオーム−cm)から入手した。酸化鉄ナノ粒子(NP)を参照によりその全体が本明細書において援用されるCorgie,et al.,Catalysis & Biocatalysis 34(5):15−20(2016)に既述のとおり調製した。これらをpH11において4℃でN2スパージ雰囲気下で保管した。
配列番号1
二機能性P450/NADPH−P450レダクターゼ[巨大菌(Bacillus megaterium)]
配列番号2
シトクロムP450 3A4アイソフォーム1[ヒト(Homo sapiens)]
配列番号3
シトクロムP450 1A2[ヒト(Homo sapiens)]
配列番号4
CYP2D6[ヒト(Homo sapiens)]
配列番号5
シトクロムP450−2E1[ヒト(Homo sapiens)]
配列番号6
シトクロムP450−2E1[ヒト(Homo sapiens)]
配列番号7
シトクロムP450、ファミリー2、サブファミリーC、ポリペプチド9[ヒト(Homo sapiens)]
Claims (64)
- 熱可塑性ポリマー及び磁性ミクロ粒子を含む磁性マクロポーラス粉末であって、磁性ナノ粒子の自己アセンブリしたメソポーラス凝集体を固定化するための形状化磁性マクロポーラス足場の付加生産(AM)のために操作可能である、磁性マクロポーラス粉末。
- 前記磁性粒子が、約50〜100μmのサイズを有する、請求項1に記載の磁性マクロポーラス粉末。
- 前記磁性粒子が、約10〜50μmのサイズを有する、請求項1に記載の磁性マクロポーラス粉末。
- 前記磁性粒子が、約5〜10μmのサイズを有する、請求項1に記載の磁性マクロポーラス粉末。
- 前記磁性粒子が、約10μmのサイズを有する、請求項1に記載の磁性マクロポーラス粉末。
- 前記磁性粒子が、約5μmのサイズを有する、請求項1に記載の磁性マクロポーラス粉末。
- 前記磁性粒子が、5μm未満のサイズを有する、請求項1に記載の磁性マクロポーラス粉末。
- 前記磁性粒子が、100μm超のサイズを有する、請求項1に記載の磁性マクロポーラス粉末。
- 約150μmの平均サイズを有する、請求項1に記載の磁性マクロポーラス粉末。
- 約75μmの平均サイズを有する、請求項1に記載の磁性マクロポーラス粉末。
- 約15μmの平均サイズを有する、請求項1に記載の磁性マクロポーラス粉末。
- 前記磁性粒子が、0〜10重量%の濃度を有する、請求項1に記載の磁性マクロポーラス粉末。
- 前記磁性粒子が、10〜50重量%の濃度を有する、請求項1に記載の磁性マクロポーラス粉末。
- 前記磁性粒子が、50〜90重量%の濃度を有する、請求項1に記載の磁性マクロポーラス粉末。
- 前記熱可塑性ポリマーが、ポリビニルアルコール(PVA)、アクリル(PMMA)、アクリロニトリルブタジエンスチレン(ABS)、ナイロン6及びナイロン12を含むポリアミド、ポリ乳酸(PLA)、ポリベンゾイミダゾール(PBI)、ポリカーボネート(PC)、ポリエーテルスルホン(PES)、ポリオキシメチレン(POM)、ポリエーテルエーテルケトン(PEEK)、ポリエーテルイミド(PEI)、ポリエチレン(PE)、ポリフェニレンオキシド(PEO)、ポリフェニレンスルフィド(PPS)、ポリプロピレン(PP)、ポリスチレン(PS)、ポリ塩化ビニル(PVC)、ポリテトラフルオロエチレン(PTFE)、コポリエステル、及びそれらの化学的機能化誘導体からなる群から選択されるポリマーを含む、請求項1に記載の磁性マクロポーラス粉末。
- 前記磁性ミクロ粒子が、マグネタイト(Fe3O4)、ヘマタイト(α−Fe2O3)、磁赤鉄鉱(γ−Fe2O3)、スピネルフェライト、天然磁石、コバルト、ニッケル、希土類、及び磁性複合体からなる群から選択される磁性材料を含む、請求項1に記載の磁性マクロポーラス粉末。
- 前記希土類が、ネオジム、ガドリニウム、シスプロシウム、サマリウム−コバルト、又はネオジム−鉄−ホウ素である、請求項16に記載の磁性マクロポーラス粉末。
- 前記磁性複合材料が、セラミック、フェライト、又はアルニコ磁石を含む、請求項16に記載の磁性マクロポーラス粉末。
- 前記熱可塑性ポリマー及び前記磁性ミクロ粒子が、化学的にブレンドされている、請求項1〜18のいずれか一項に記載の磁性マクロポーラス粉末。
- 前記熱可塑性ポリマー及び前記磁性ミクロ粒子が、熱的にブレンドされている、請求項1〜18のいずれか一項に記載の磁性マクロポーラス粉末。
- 前記熱可塑性ポリマー及び前記磁性ミクロ粒子が、物理的にブレンドされている、請求項1〜18のいずれか一項に記載の磁性マクロポーラス粉末。
- 0.5〜200μmのサイズを有するマクロ細孔を含む、請求項1〜21のいずれか一項に記載の磁性マクロポーラス粉末。
- セルロース繊維、セルロースナノファイバー、ガラス繊維、又は炭素繊維をさらに含む、請求項1〜22のいずれか一項に記載の磁性マクロポーラス粉末。
- 磁性ナノ粒子の自己アセンブリしたメソポーラス凝集体及び前記メソ細孔内に又はそれらの表面上に磁気的に固定化された酵素をさらに含む、請求項1〜23のいずれか一項に記載の磁性マクロポーラス粉末。
- 請求項1〜23のいずれか一項に記載の磁性マクロポーラス粉末を含み、前記粉末は、三次元(3D)プリンティングにより前記形状に形成された、形状化磁性マクロポーラス足場。
- 前記形状が、円柱、球体、ビーズ、ストリップ、カプセル、立方体、スクエアロッド、ピラミッド、ダイヤモンド、格子、又は不規則形状である、請求項25に記載の形状化磁性マクロポーラス足場。
- 磁性ナノ粒子の自己アセンブリしたメソポーラス凝集体をさらに含む、請求項25又は26に記載の形状化磁性マクロポーラス足場。
- 磁性ナノ粒子の前記自己アセンブリしたメソポーラス凝集体が、前記磁性ナノ粒子の前記メソ細孔内に又は表面上に磁気的に固定化された1つ以上の酵素をさらに含む、請求項27に記載の形状化磁性マクロポーラス足場。
- 前記1つ以上の酵素が、ヒドロラーゼ、ヒドロキシラーゼ、過酸化水素生成酵素(HPP)、ニトララーゼ、ヒドラターゼ、脱水素酵素、トランスアミナーゼ、ケトレダクターゼ(KREDS)エネレダクターゼ(EREDS)、イミンレダクターゼ(IREDS)、カタラーゼ、ジスムターゼ、オキシダーゼ、ジオキシゲナーゼ、リポキシダーゼ、オキシドレダクターゼ、ペルオキシダーゼ、ラッカーゼ、シンテターゼ、トランスフェラーゼ、オキシニトリラーゼ、イソメラーゼ、グルドシダーゼ、キナーゼ、リアーゼ、スクラーゼ、インベルターゼ、エピメラーゼ、及びリパーゼからなる群から選択される、請求項28に記載の形状化磁性マクロポーラス足場。
- 磁性ナノ粒子の前記自己アセンブリしたメソポーラス凝集体が、ミクロソームを含み、第1の酵素活性を有する拡散性補助因子を要求する第1の酵素は、前記ミクロソーム内に含有され、補助因子再生活性を含む第2の酵素は、前記メソ細孔内に磁気的に包括され、前記補助因子は、前記第1の酵素活性において利用され;前記第1及び第2の酵素は、拡散性基質を拡散性生成物に変換することにより機能し;前記磁性ナノ粒子は、前記磁性マクロポーラス足場と磁気的に会合している、請求項27に記載の形状化磁性マクロポーラス足場。
- 磁性ナノ粒子の前記自己アセンブリしたメソポーラス凝集体が、第1の酵素活性を有する拡散性補助因子を要求する第1の酵素;補助因子再生活性を含む第2の酵素を含み、前記補助因子は、前記第1の酵素活性において利用され;前記第1及び第2の酵素は、磁性ナノ粒子の前記凝集体により形成された前記メソ細孔内に磁気的に包括され、前記第1及び第2の酵素は、拡散性基質を拡散性生成物に変換することにより機能する、請求項27に記載の形状化磁性マクロポーラス足場。
- 前記第1の酵素が、酸化酵素である、請求項31に記載の形状化磁性マクロポーラス足場。
- 前記酸化酵素が、フラビン含有オキシゲナーゼであり;前記組成物が、前記第1の酵素と同時局在する補助因子レダクターゼ活性を有する第3の酵素をさらに含む、請求項32に記載の形状化磁性マクロポーラス足場。
- 前記酸化酵素が、P450モノオキシゲナーゼであり;前記組成物が、前記第1の酵素と同時局在する補助因子レダクターゼ活性を有する第3の酵素をさらに含む、請求項32に記載の形状化磁性マクロポーラス足場。
- 前記P450モノオキシゲナーゼ及び前記第3の酵素が、単一タンパク質内に含まれる、請求項32に記載の形状化磁性マクロポーラス足場。
- 前記単一タンパク質が、二機能性シトクロムP450/NADPH−−P450レダクターゼを含む、請求項35に記載の形状化磁性マクロポーラス足場。
- 前記単一タンパク質が、BM3活性を有し、配列番号1との少なくとも90%の配列同一性を有する、請求項35に記載の形状化磁性マクロポーラス足場。
- 前記足場が、特定の生体触媒プロセスに適した形状に形成される、請求項25〜37のいずれか一項に記載の形状化磁性マクロポーラス足場。
- 請求項1〜23のいずれか一項に記載の磁性マクロポーラス粉末を含む形状化磁性マクロポーラス足場を形成する方法であって、三次元(3D)プリンタを用いて前記形状化磁性マクロポーラス足場を付加生産(AM)し、前記形状を3Dモデルから得るステップを含む方法。
- 前記3Dモデルが、電子ファイルである、請求項39に記載の方法。
- 前記電子ファイルが、コンピュータ支援設計(CAD)又はステレオリソグラフィー(STL)ファイルである、請求項40に記載の方法。
- 前記AMが、熱融解フィラメント製法(FFF)又は選択的レーザ焼結(SLS)である、請求項39〜41のいずれか一項に記載の方法。
- 前記マクロ細孔が、塩、糖、又は小型可溶性ポリマーからなる群から選択される可溶性薬剤を用い、前記可溶性薬剤を溶剤により除去して形成される、請求項39〜42のいずれか一項に記載の方法。
- 酵素反応を触媒するための装置を形成する方法であって、形状化磁性マクロポーラス足場を、磁性ナノ粒子の自己アセンブリしたメソポーラス凝集体及び酵素と組み合わせ、前記酵素は、前記メソ細孔内に磁気的に固定化されるステップを含む方法。
- 複数の基質間の反応を触媒する方法であって、請求項24に記載の磁性マクロポーラス粉末を、前記酵素が前記基質間の前記反応を触媒する条件下で前記基質に露出させるステップを含む方法。
- 複数の基質間の反応を触媒する方法であって、請求項28又は29に記載の形状化磁性マクロポーラス足場を、前記酵素が前記基質間の前記反応を触媒する条件下で前記基質に露出させるステップを含む方法。
- 前記反応が、医薬製品の生産において用いられる、請求項45又は46に記載の方法。
- 前記反応が、医薬品の生産に用いられる、請求項45又は46に記載の方法。
- 前記反応が、食品製品の生産に用いられる、請求項45又は46に記載の方法。
- 前記反応が、風味料の生産に用いられる、請求項45又は46に記載の方法。
- 前記反応が、香料の生産において用いられる、請求項45又は46に記載の方法。
- 前記反応が、甘味料の生産において用いられる、請求項45又は46に記載の方法。
- 前記反応が、農薬の生産において用いられる、請求項45又は46に記載の方法。
- 前記反応が、抗菌剤の生産において用いられる、請求項45又は46に記載の方法。
- 前記反応が、毒素の生産において用いられる、請求項45又は46に記載の方法。
- 前記反応が、洗剤の生産において用いられる、請求項45又は46に記載の方法。
- 前記反応が、燃料製品の生産に用いられる、請求項45又は46に記載の方法。
- 前記反応が、生化学製品の生産に用いられる、請求項45又は46に記載の方法。
- 前記反応が、紙製品の生産に用いられる、請求項45又は46に記載の方法。
- 前記反応が、プラスチック製品の生産に用いられる、請求項45又は46に記載の方法。
- 前記反応が、溶液から汚染物を除去するプロセスにおいて用いられる、請求項45又は46に記載の方法。
- 前記溶液は、水溶液、溶剤、又は油である、請求項61に記載の方法。
- 前記メソポーラス凝集体を除去し、それらをメソポーラス凝集体の新たな調製物により置き換えるステップをさらに含む、請求項45又は46に記載の方法。
- フローセル及び連続生産を使用して実施される、請求項45又は46に記載の方法。
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CN109068659A (zh) * | 2016-04-16 | 2018-12-21 | 齐姆特罗尼克斯催化系统股份有限公司 | 用于固定生物纳米催化剂的磁性大孔聚合物杂化支架 |
US10135306B2 (en) * | 2016-07-14 | 2018-11-20 | National Cheng Kung University | Reluctance motor and flux barrier structure thereof |
EP3548175A4 (en) * | 2016-12-03 | 2020-08-05 | Zymtronix Catalytic Systems, Inc. | MAGNETICALLY IMMOBILIZED METABOLIC ENZYMES AND COFACTOR SYSTEMS |
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2019
- 2019-09-26 EP EP19867569.6A patent/EP3856900A4/en active Pending
- 2019-09-26 CN CN201980063035.0A patent/CN113166749A/zh active Pending
- 2019-09-26 CA CA3114353A patent/CA3114353A1/en active Pending
- 2019-09-26 JP JP2021517462A patent/JP2022500067A/ja active Pending
- 2019-09-26 WO PCT/US2019/053307 patent/WO2020069227A1/en unknown
- 2019-09-26 US US17/274,164 patent/US20210189374A1/en active Pending
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EP3856900A4 (en) | 2022-06-29 |
CA3114353A1 (en) | 2020-04-02 |
WO2020069227A1 (en) | 2020-04-02 |
US20210189374A1 (en) | 2021-06-24 |
CN113166749A (zh) | 2021-07-23 |
EP3856900A1 (en) | 2021-08-04 |
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