中文摘要
本論文研究開發出另一種促進silica沉澱的方式，在酸水解後的tetramethylorthosilicate (TMOS)中加入酸水解後的3-aminopropyl triethoxysilane(APTS)及酵素溶液會促成silica沉澱，在silica沉澱的過程中酵素會被包埋在silica顆粒中而形成固定化酵素。經電子顯微鏡(SEM)觀察silica顆粒粒徑約為400~500nm，在包埋固定化牛血清蛋白時，最適固定化的條件為pH7~9的Tris-HCl緩衝液，牛血清蛋白濃度為2.5mg/mL，可全部被包埋。
D-p-hydroxyphenylglycine( D-p-HPG )為抗生素Amoxicillin的前驅物之一，而D-hydantoinase為製備D-p-HPG的必要酵素。因此本論文以基因重組技術建構從Agrobacterium radiobacter來的D-hydantoinase表現質體，在E. coli BL21(DE)中大量表現，在直接固定化D-hydantoinase粗萃液時，每克silica乾重約可包埋189.3mg的蛋白質，包埋率約為82.3%，活性回收率約為91.86%，silica顆粒粒徑約為50~80nm。因silica顆粒在pH>8的環境中會被水解，在經戊二醛表面處理後可達到保護silica效果，防止酵素漏失，其活性回收率約為71.32%，最適戊二醛濃度為2%，酵素最適反應溫度為55℃~60℃，在55℃下，固定化後可使半生期增加3倍，固定化後之酵素重複使用七次後，殘餘活性仍可達92%。

ABSTRACT
D-hydantoinase, encoded from recombinant Escherichia coli BL21 (DE3) harbouring plasmid pET30b derived from Agrobacterium radiobacter, was used as an immobilization enzyme to catalyst the production of N-carbamoyl-D-phenylglycine from DL-p-phenylhydantoin, an intermediate substance for the production of D-phenylglycine. The encapsulation of enzyme is an attractive approach to prevent the enzyme from leakage. Enzyme-encapsulated in silica is the commenly used and the matrix is usually formed by sol-gel processing, by using polypeptides (polyamines), or biomimicking catalysts, i.e. cysteamine, to initiate silica polycondensation. In this study, silica matrix are formed from silicic acid by acid hydrolyzed of 3-aminopropyl triethoxysilane (APTS) under neutral pH and at ambient temperature. It was observed by scanning electron microscopy (SEM) that the diameter of silica spheres formed after silification is approximately 500 nm. BSA is used as test protein to be immobilized into the silica before D-hydantoinase immobilization is taken place. It was shown that BSA concentration as high as 2.5mg/mL could be totally encapsulation. D-hydantoinase of 189.3 mg was immobilized in one gram of dry silica matrix and the efficiency of encapsulated D-hydantoinase in silica matrix was 82.3%. As compared to its free enzyme, its activity was 91.86% and the half-life was 3 times longer at 55 oC. However, upon extended operation the enzyme catalytic activity was decreased significantly due to enzyme leakage and the silica matrix was degraded due to alkaline condition applied during the enzymatic reaction. Glutaraldehyde was then used to form a protection film covering the silica matrix by a reaction with the amine group on silica which is derived from APTS. The enzyme activity yield was reduced to 70% after glutaraldehyde modification and its half life was 3.5 times longer at 55 oC than free enzyme. Moreover, immobilized D-hydantoinase could be reused for 7 times to maintain 92% residual activity. The enzymatic reaction followed Michaelis-Menten equation and the kinetics of the enzyme will be discussed.

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