| Summary: | 碩士 === 國立臺灣科技大學 === 化學工程系 === 96 === 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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