| Summary: | 碩士 === 國立清華大學 === 生物技術研究所 === 87 === The N-carbamoyl-D-amino-acid amidohydrolase (Nca) is an important enzyme that catalyzes the hydrolysis of N-carbamoyl-D-p-hydroxyphenylglycine (Nca-HPG) to D-hydroxyphenylglycine (D-p-HPG). The nca gene of Agrobacterium radiobacter DH101 was cloned into vector pQE-30 (pQE-NCA) and expressed in Escherichia coli cells. We used His6-tag technology for easy purification of the recombinant proteins and attempted to improve Nca on the stability of the immobilized enzyme in repeated reactions.
E. coli JM109 (pQE-NCA) was cultivated at 37℃, and then induced with IPTG at 25℃ for 17 hours, which can harvested soluble Nca protein. After purification by affinity column chromatography, the Nca of amounted to 8 mg per 200 ml culture medium. The various support matrices for immobilization were screened, the resin DEAE Sepharose CL-6B had higher adsorbed Nca activity in repeated reactions. The optimal pH and ion strength of the free Nca and the immobilized Nca were determined to be about 7.0 and 120 mM potassium phosphate buffer. The metal ions Cu2+ and Hg2+ inhibited the activity of the free Nca and the immobilized Nca, but Zn2+ only inhibited the free Nca. After 4 times repeated reactions, the remaining activity of the immobilized enzyme with 150 mM potassium phosphate buffer in the reaction mixture was 10%. After 7 times repeated reactions, with 150 mM potassium phosphate buffer and cross-linked with 0.2% glutaraldehyde, the remaining activity was 9%. And after 14 times repeated reactions, with 150 mM potassium phosphate、0.2% glutaraldehyde and contain dithiothreitol, sealed under nitrogen, the remaining activity of the immobilized enzyme was 62%.
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