| 摘要: | 本研究之主要目的在探討具備巨大孔徑幾丁聚醣膜的製備並藉由薄膜上吸附性重金屬的摻合來改變薄膜表面官能基特性，再將其應用於固定具特定生化功能的酵素。本研究特別針對牛血清蛋白，胰蛋白酶、尿素酶，木瓜蛋白酶以及酪蛋白酶等幾種於生化和環境污染物降解上應用廣泛的酵素進行固定化應用研究。幾丁聚醣作為固定基材之前其表面必須先以具環氧基(epoxide groups)之高分子化合物(例如1, 4 butanediol diglycidyl ether, glutaraldehyde and epichlorohydrin) 進行塗佈以使幾丁聚醣膜表面功能化(活化)(surface-functionalized)。幾丁聚醣膜表面功能化主要係利用“光誘發„接枝的共聚合效應，將對酵素具高親和力的受體或能與其共價鍵結的官能基經反應鍵結於聚醣膜表面。固定化條件、阻斷成因、洗滌步驟和儲存條件之最佳化為本計劃重要研究主題之ㄧ；同時，經活化膜表面所固定酵素的數量以及固定化酵素的活性，亦被列為重要探討因子。製備這種嶄新具固定化酵素的多醣體薄膜，許多因子均會影響酵素固定膜之表面特性及其後續實際應用性。但是，其中首要克服之影響條件為巨大孔徑幾丁聚醣膜的製備，此多醣體膜必須有別於傳統多孔性吸附介質以便於酵素的固定僅止於表面而不至於造成過多酵素在孔隙間擴散。多孔性基材的擴散性會影響固定化酵素的活性、催化效率及再利用性。本研究預計以矽膠為固定支持物並利用相轉化製備方法將幾丁聚醣交聯偶合於矽膠表面以形成具備巨大孔徑的幾丁聚醣薄膜。此多醣體薄膜經由表面“功能化„後可針對特定酵素予以共價或配位鍵結使其固定於薄膜表面。至於本研究所合成的固定化酵素/幾丁聚醣膜的實際應用雖然不是本計劃之研究重點，但將朝biomedical, bioenginerring 及bioanalytical 等領域進行初步的探討，目前較為具體的應用標的為土壤有機污染物與廢水生物處理的 Bioremediation，尤其是，食品、製酒與飲料業之廢水生物處理為首要探討標的。 The present proposal is aimed at to investigate the preparation of macroporous chitosan membrane and attached metal ions incorporated membrane microspheres form for immobilization of a model enzyme (biofunctionalized enzyme). In particular, the covalent immobilization of enzymes (BSA, Trypsin, Urease, papain, tyrosin, Aminotrans ferase) on macroporous chitosan membrane with a nominal cut-off of about 0.02 to 0.4 μm which had been surface-functionalized with a thin polymer layer with reactive epoxide groups (1, 4 butanediol diglycidyl ether, glutaraldehyde and epichlorohydrin). The surface functionalization is based on a photo-initiated graft copolymerization, which has optimized for the preparation of affinity membranes for covalent immobilizations of proteins as affinity receptors. The optimization of the immobilization protocol (immobilization conditions, blocking and washing steps, storage conditions) is another topic. The resulting enzyme-membranes shall be characterized in terms of amount of immobilized enzyme and enzyme activity will be investigated. To prepare a novel immobilized biofunctional membrane evaluate of their properties and application by reversible enzyme immobilization. These support systems could provide a large specific surface area and therefore, the enzyme can be immobilized only on the surface, and would also provide less diffusion limitation problem than that of the porous supports. The resultant immobilized enzyme systems can be characterized and their activity retention, immobilization efficiency, catalytic properties, reusability and stability aspects are compared. The expected results of the present research are silica gel supported cross-linked chitosan membrane with surface macropores can be prepared by phase inversion methods. This silica gel in the core of the membrane could act as a rigid support and the macroporous chitosan layer on the bead surface could provide a sufficient amount of amino groups and suitable steric position for enzyme coupling. The product of this research, the macroporous chitosan biofunctionalized membrane could find many promise applications in biomedical, bioengineering and bioanalytical fields. Besides that, the protocol of relating enzyme immobilization method to its structure might lead to the rational design of enzyme immobilization considering the specificity of the molecular. They include synthetic and analytical purposes, bioremediation of contaminated soils and wastewater treatment. Among them, wastewater treatment, and wine and beverages stabilization appear to be very promising and, with regard to this aspect, it is worthwhile to make some considerations 研究期間：10003 ~ 10102 |
