A cDNA clone of 1114 bp encoding a putative Mn-superoxide dismutase (Mn-SOD) from diatom
Thallassiosira 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 from
E. 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 analka
line 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)