碩士 / 國立海洋大學 / 水產生物技術研究所 / 89 / Abstract
A full-length cDNA clone of 774 bp encoding a putative copper / zinc-superoxide dismutase ( Cu/Zn-SOD ) from red porgy (Pagrus major ) was cloned by PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete epenreading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (53~91%) with the sequences of the Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper ( His-47, 49, 64 and 121 ) and zinc ( His-64, 72, 81 and Asp-84 ), as well as the two cysteines ( 58 and 147 ) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the red porgy Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b (+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native gel and purified by Ni2+- nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The enzyme retained about 75% activity after heating 70 ℃for 10 min. The half-life was 15 min and the inactivation rate constant (Kd) was 9.64×10-2 min-1 at 70℃. About 30% protein of the enzyme was dissociated into monomer from under 4% SDS or 1M imidazole. The enzyme was much more resistant to trypsin attack than chymotrypsin.
| Identifer | oai:union.ndltd.org:TW/089NTOU0613010 |
| Date | January 2001 |
| Creators | De-Feng Weng, 翁得峰 |
| Contributors | Chi-Tasi Lin, 林棋財 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 0 |
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