碩士 / 國立海洋大學 / 水產生物技術研究所 / 90 / Abstract
A full-length cDNA with 1100 bp encoding a putative Manganese-superoxide dismutase ( Mn-SOD ) from diatoms ( Thallassiosira weissflogii ) was cloned. Nucleotide sequence analysis indicated that it comprises a complete open reading frame coding for 218 amino acid residues. The deduced amino acid sequence showed high similarity (49 ~ 65%) with that of Mn-SOD from other species. Computer analysis of the residues required for coordinating the single trivalent manganese iron and the 11 residues putatively involved in the active center were well conserved among all reported Mn-SOD sequence. To further characterize the diatoms Mn-SOD, the coding region was subcloned into an expression vector, pET-20b (+), and transformed into Escherichia coli BL21(DE3)pLysS. The expression of the Mn-SOD was confirmed by enzyme activity stained on a Native gel and purified by Ni2+- nitrilotriacetic acid Sepharose superflow. The diatoms Mn-SOD enzyme retained about 88% activity after heating 60℃for 10 min. The half-life was 14.7 min and the inactivation rate constant (Kd) was 3.21×10-2 min-1 at 65℃. The enzyme was inhibited under acidic pH (below 5.0) and 1.6M imidazole. About 23% protein of the enzyme was dissociated under 4% SDS and the enzyme was much more resistant to trypsin attack than chymotrypsin.
As above, A full-length cDNA with 1120 bp encoding a putative Iron-superoxide dismutase ( Fe-SOD ) from diatoms ( Thallassiosira weissflogii )was cloned. Nucleotide sequence analysis indicated that it comprises a complete open reading frame coding for 202 amino acid residues. The deduced amino acid sequence showed high similarity (49 ~ 68%) with that of Fe-SOD from other species. Computer analysis of the residues required for coordinating the single trivalent iron ion and the 11 residues putatively involved in the active center were well conserved among all reported Fe-SOD sequence. To further characterize the diatoms Fe-SOD, the coding region was subcloned into an expression vector, pET-20b (+), and transformed into Escherichia coli BL21(DE3)pLysS. The expression of the Fe-SOD was confirmed by enzyme activity stained on a Native gel and purified by Ni2+- nitrilotriacetic acid Sepharose superflow. The diatoms Fe-SOD enzyme retained about 83% activity after heating 50℃for 10 min. The half-life was 23 min and the inactivation rate constant (Kd) was 3.03×10-2 min-1 at 55℃. The enzyme was inhibited under acidic pH (below 3.0) and 1.6M imidazole. About 27% protein of the enzyme was dissociated under 4% SDS and the enzyme was much more resistant to trypsin attack than chymotrypsin.
| Identifer | oai:union.ndltd.org:TW/090NTOU0613009 |
| Date | January 2002 |
| Creators | 黃宗賢 |
| Contributors | 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 | 145 |
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