研究生: |
張名棻 Ming-Fen Chang |
---|---|
論文名稱: |
磁性矽化包埋 β-葡萄糖苷酶之研究 Silicification of β-glucosidase for its immobilization on magnetic nanoparticle surface. |
指導教授: |
李振綱
Cheng-Kang Lee |
口試委員: |
陳秀美
Hsiu-Mei Chen 王勝仕 Steven S.-S. Wang |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 化學工程系 Department of Chemical Engineering |
論文出版年: | 2013 |
畢業學年度: | 101 |
語文別: | 中文 |
論文頁數: | 104 |
中文關鍵詞: | 矽化 、包埋 、β-葡萄糖苷酶 、磁珠 |
外文關鍵詞: | silicification, magnetic nanoparticles, β-Glucosidase, immobilization |
相關次數: | 點閱:270 下載:2 |
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本論文研究是以仿生物矽化的方式對蛋白質進行包埋,進而達到穩定蛋白質結構,使蛋白質在較為不利的環境下,仍能維持較高的活性,為利於包埋之蛋白質回收再使用,在矽化過程中將混入奈米磁粒,製備成具磁性之矽化蛋白質。聚乙烯亞胺( Polyethylenimine, PEI),是一個帶有高密度正電荷的有機大分子,與矽藻中具有聚合矽酸功能的Silaffin蛋白功能相同,能將矽酸分子聚合成顆粒。
本論文先合成表面具有PEI的奈米磁粒(40~50nm),再與β-葡萄糖苷酶以靜電作用方式結合,之後以矽酸溶液進行矽化包埋。β-葡萄糖苷酶,又稱為 β-D-glucosidase 或cellobiase,主要會對纖維雙醣進行水解,產生葡萄糖單體,固定化後的β-葡萄糖苷酶活性回收率約為91.23%,最適反應PH為4~4.5,最適反應溫度為55℃~60℃,浸入60℃下2.5小時,固定化後的β-葡萄糖苷酶仍維持51.13%的活性,自由態酵素則完全失去活性;以矽化包埋固定化之β-葡萄糖苷酶,重複使用5次後,殘餘活性仍可達69.8%。此磁性矽化之β-葡萄糖苷酶可應用於纖維素之酵素水解上,減輕纖維寡糖抑制纖維素水解酶的問題,提升纖維素之水解速率。
β-Glucosidase, from Aspergillus niger, was used as a model enzyme to be immobilized by silicification on magnetic nanoparticles. The enzyme catalysts the reaction of converting the colorless substrate p-nitrophenyl-β-D- glucopyranoside (p-NPG) to yellowish production p-nitrophenol. Enzymes immobilization by encapsulation in silica have been studied for some times but usually carried out by solvent containing sol-gel process. Recently, polycationic peptides isolated from diatom cell walls such as R5 have been used to induce the precipitation of silicic acid in the presence of enzyme for the purpose of enzyme immobilization in silica. In addition to polycationic peptides, polycationic compound such as polyethyleneimine (PEI) was found to be a good biomimicking catalyst can also induce the polycondensation of silicic acid. In this thesis, silicic acids from hydrolyzed tetramethyl orthosilicate (TMOS) and neutralized sodium silicate were employed to be precipitated by PEI coated on β-glucosidase immobilized magnetic nanoparticles (MPBP) which was prepared by immobilizing β-glucosidase on PEI coated magnetic nanoparticles (MP) via electrostatic interactions. The size of silicificated β-glucosidase magnetic particles is approximately 350 nm with β-glucosidase content about 4.3 mg/gram of dry MP. As compared with free enzyme, the relative activity of silicificated β-glucosidase is 91.84% with half-life about 4 times longer at 60℃. Moreover, immobilized β-glucosidase can be reused for 5 times still maintains 70% of its initial activity. The facile enzyme immobilization method demonstrated that silica encapsulation can protect the β-glucosidase from leakage and denaturation, and the β-glucosidase activity can be easily retrieved by applying a magnetic field.
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