Characterization of Fe/Mn-Superoxide Dismutase from Diatom Thallassiosira weissflogii: Cloning, Expression, and Property
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- 作者：Chuian-Fu Ken ; Tung-Ming Hsiung ; Zong-Xian Huang ; Rong-Huay Juang ; and Chi-Tsai Lin
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2005
- 出版时间：March 9, 2005
- 年：2005
- 卷：53
- 期：5
- 页码：1470 - 1474
- 全文大小：256K
- 年卷期：v.53,no.5(March 9, 2005)
- ISSN：1520-5118
文摘
A cDNA clone of 1114 bp encoding a putative Mn-superoxide dismutase (Mn-SOD) from diatomThallassiosira weissflogii was cloned by the PCR technique. Nucleotide sequence analysis of thiscDNA clone revealed that it was translated into 201 amino acid residues. When the sequence wascompared with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs fromE. coli and Photobacterium leiognathi, this SOD showed higher homology to Mn-SOD. The aminoacid residues required to coordinate the single manganese ion were conserved in all reported Mn-SOD sequences. This cDNA was introduced in an expression vector, pET-20b(+), and transformedinto E. coli BL21(DE3)pLysS. The expressed SOD protein was then purified by a His-tag column.The recombinant enzyme was heated at 55 
C with a time-dependent assay; the time interval for50% inactivation was 23 min, and its thermal inactivation rate constant Kd was 3.03 × 10-2 min-1.The enzyme was inactivated either in acidic pH (below 4.0) or in the presence of imidazole (above1.6 M) and had only a moderate effect under SDS (above 4%), whereas it was not affected under analkaline pH (above 9.0). The atomic absorption spectrometric assay showed that 0.6 atom of iron/manganese (3:1) was present in each subunit of SOD. Reconstitution study was suggested thatdiatom SOD was cambialistic (Fe/Mn)-SOD. The finding of this SOD cDNA could be used for areference in comparing the differences among marine phytoplankton species and as a probe to detectthe transcription level of this enzyme, which can be applied in cosmetics for skin protection or defendingunesthetic effects caused by oxygen-containing free radicals.Keywords: Diatom; Thallassiosira weissflogii; expression; cambialistic-superoxide dismutase(Fe/Mn-SOD)
C with a time-dependent assay; the time interval for50% inactivation was 23 min, and its thermal inactivation rate constant Kd was 3.03 × 10-2 min-1.The enzyme was inactivated either in acidic pH (below 4.0) or in the presence of imidazole (above1.6 M) and had only a moderate effect under SDS (above 4%), whereas it was not affected under analkaline pH (above 9.0). The atomic absorption spectrometric assay showed that 0.6 atom of iron/manganese (3:1) was present in each subunit of SOD. Reconstitution study was suggested thatdiatom SOD was cambialistic (Fe/Mn)-SOD. The finding of this SOD cDNA could be used for areference in comparing the differences among marine phytoplankton species and as a probe to detectthe transcription level of this enzyme, which can be applied in cosmetics for skin protection or defendingunesthetic effects caused by oxygen-containing free radicals.Keywords: Diatom; Thallassiosira weissflogii; expression; cambialistic-superoxide dismutase(Fe/Mn-SOD)