- 學位論文
以聚胺催化仿生氧化矽包覆D型胺基酸氧化酶及其最適化
Encapsulation of D-amino acid oxidase from Rhodospo-ridium toruloides in polyamine-catalyzed biomimetic silica and its optimization
摘要
利用生物矽化進行酵素固定化為新興之研究領域，此類固定化方式反應條件溫和且須時甚短。目前以對矽藻Cylindrotheca fusiformis氧化矽細胞壁形成機制之研究最為詳盡，其形成與帶有高度後修飾蛋白質silaffin有關。於in vitro實驗中，生物矽化顆粒可利用silaffin、R5胜肽(未修飾之silaffin組成片段)、甚至聚胺如polyallylamine (PAA)、polyethyleneimine (PEI)與矽酸反應形成。本研究利用PAA及PEI催化矽酸包覆RtDAO (D-amino acid oxidase from Rhodosporidum toruloides)及magnetic nanoparticle (MNP)，探討不同因子之影響。對包覆相RtDAO活性及包覆率的影響，以RtDAO濃度、MNP濃度及polyethylene glycol (PEG)濃度最顯著。以反應曲面法(response surface methodology, RSM)實驗設計對前述因子進行最適化，以PAA及PEI催化矽酸包覆RtDAO經此條件包覆之酵素活性約分別為未最適化之包覆相酵素的7及3倍。於熱穩定性實驗中，液相RtDAO其Tm為44℃。以PAA催化氧化矽包覆RtDAO，未經最適化及經RSM最適化其Tm分別為57.9℃及47.9℃；以PEI催化氧化矽包覆RtDAO，未經最適化及經RSM最適化其Tm分別為55.5℃及45.4℃。以PAA或PEI催化矽酸包覆RtDAO，在未經RSM最適化前，其熱穩定性明顯改善；但經RSM最適化後，熱穩定性則無明顯提升。
並列摘要
The application of biosilicification for enzyme immobilization has been an active area of research; it offers advantages such as mild reaction conditions and short reaction time. The cell wall formation of diatom Cylindrotheca fusiformis has been extensively studied; the cell wall formation was triggered by a highly post-translationally modified protein called silaffin. Biomimetic silica particles can be synthesized in vitro by reacting silicic acid with silaffin, or synthetic R5 peptide (one of the silaffin repeating units), or even polyamines such as polyallylamine (PAA) and polyethyleneimine (PEI). In this work, RtDAO (D-amino acid oxidase from Rhodosporidum toruloides) and magnetic nanoparticle (MNP) were encapsulated within PAA- or PEI-catalyzed biomimetic silica. The concentrations of RtDAO, MNP, and polyethylene glycol affected the activity of encapsulated RtDAO and encapsulation efficiency significantly. Using response surface methodology (RSM) to optimize the three previously mentioned factors, for PAA- and PEI-catalyzed biomimetic silica, the activity of encapsulated RtDAO was enhanced by 7.5 and 3 fold, respectively. From thermal stability study, the Tm value of free RtDAO was 44℃. For PAA-catalyzed biomimetic silica, the Tm values were, before and after optimization, 57.9℃ and 47.9℃, respectively. For PEI-catalyzed biomimetic silica, the Tm values were, before and after optimization, 55.5℃ and 45.5℃, respectively. Before optimization using RSM, the thermal stability of RtDAO was improved in PAA- or PEI-catalyzed biomimetic silica; however, similar effect was not observed after opti-mization using RSM.