Characterization of Fe/Mn-superoxide dismutase from diatom Thallassiosira weissflogii: Cloning, expression, and property

Chuian Fu Ken, Tung Ming Hsiung, Zong Xian Huang, Rong Huay Juang, Chi Tsai Lin

Research output: Contribution to journalArticle

68 Citations (Scopus)

Abstract

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 this cDNA clone revealed that it was translated into 201 amino acid residues. When the sequence was compared 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 amino acid 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 transformed into 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 for 50% 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 (above 1.6 M) and had only a moderate effect under SDS (above 4%), whereas it was not affected under an alkaline 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 that diatom SOD was cambialistic (Fe/Mn)-SOD. The finding of this SOD cDNA could be used for a reference in comparing the differences among marine phytoplankton species and as a probe to detect the transcription level of this enzyme, which can be applied in cosmetics for skin protection or defending unesthetic effects caused by oxygen-containing free radicals.

Original languageEnglish
Pages (from-to)1470-1474
Number of pages5
JournalJournal of Agricultural and Food Chemistry
Volume53
Issue number5
DOIs
Publication statusPublished - 2005 Mar 9

Fingerprint

Diatoms
Bacillariophyceae
Cloning
Superoxide Dismutase
Organism Cloning
molecular cloning
superoxide dismutase
Complementary DNA
Escherichia coli
Manganese
Vibrio mimicus
Assays
Enzymes
Clone Cells
Photobacterium
Amino Acids
Phytoplankton
Cosmetics
Transcription
Free Radicals

All Science Journal Classification (ASJC) codes

  • Chemistry(all)
  • Agricultural and Biological Sciences(all)

Cite this

Ken, Chuian Fu ; Hsiung, Tung Ming ; Huang, Zong Xian ; Juang, Rong Huay ; Lin, Chi Tsai. / Characterization of Fe/Mn-superoxide dismutase from diatom Thallassiosira weissflogii : Cloning, expression, and property. In: Journal of Agricultural and Food Chemistry. 2005 ; Vol. 53, No. 5. pp. 1470-1474.
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abstract = "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 this cDNA clone revealed that it was translated into 201 amino acid residues. When the sequence was compared 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 amino acid 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 transformed into 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 for 50{\%} 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 (above 1.6 M) and had only a moderate effect under SDS (above 4{\%}), whereas it was not affected under an alkaline 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 that diatom SOD was cambialistic (Fe/Mn)-SOD. The finding of this SOD cDNA could be used for a reference in comparing the differences among marine phytoplankton species and as a probe to detect the transcription level of this enzyme, which can be applied in cosmetics for skin protection or defending unesthetic effects caused by oxygen-containing free radicals.",
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Characterization of Fe/Mn-superoxide dismutase from diatom Thallassiosira weissflogii : Cloning, expression, and property. / Ken, Chuian Fu; Hsiung, Tung Ming; Huang, Zong Xian; Juang, Rong Huay; Lin, Chi Tsai.

In: Journal of Agricultural and Food Chemistry, Vol. 53, No. 5, 09.03.2005, p. 1470-1474.

Research output: Contribution to journalArticle

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T1 - Characterization of Fe/Mn-superoxide dismutase from diatom Thallassiosira weissflogii

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AU - Ken, Chuian Fu

AU - Hsiung, Tung Ming

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AU - Juang, Rong Huay

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AB - 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 this cDNA clone revealed that it was translated into 201 amino acid residues. When the sequence was compared 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 amino acid 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 transformed into 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 for 50% 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 (above 1.6 M) and had only a moderate effect under SDS (above 4%), whereas it was not affected under an alkaline 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 that diatom SOD was cambialistic (Fe/Mn)-SOD. The finding of this SOD cDNA could be used for a reference in comparing the differences among marine phytoplankton species and as a probe to detect the transcription level of this enzyme, which can be applied in cosmetics for skin protection or defending unesthetic effects caused by oxygen-containing free radicals.

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